ES2795926T3 - Procedure to obtain immunostimulating dendritic cells - Google Patents

Abstract

A method for inducing the differentiation of monocytes contained in an extracorporeal amount of a blood sample from a mammalian subject into immunostimulating autologous dendritic cells, said method comprising at least the steps of: a) subjecting said extracorporeal amount of said blood sample from a mammalian subject at a shear voltage passing through a flow chamber that allows a fixed or adjustable flow rate adjustment; b) wherein the procedure is performed in the absence of photoactivatable agents such as 8-MOP and UVA c) wherein the procedure is performed without the need to add a molecular cocktail comprising cytokines; and d) in which the differentiation of monocytes from said blood sample into immunostimulatory autologous dendritic cells is identified by the increased expression of at least one molecular marker HLA-DR, CD83, CD86, ICAM1 or PLAUR and no increased expression of GILZ upon comparing immunostimulatory autologous dendritic cells with monocytes within the extracorporeal amount of a mammalian subject blood sample.

Description

DESCRIPTION

Procedure to obtain immunostimulating dendritic cells

FIELD OF THE INVENTION

The present invention relates to processes for producing immunostimulatory dendritic cells. The present invention in particular relates to a method of preferential production of immunostimulatory dendritic cells over immunosuppressive dendritic cells.

BACKGROUND OF THE INVENTION

Dendritic cells (DC) are recognized as potent antigen presenting cells for initiating and controlling cellular immune responses in humans. Since DCs can be immunostimulatory or immunosuppressive, depending on which set of their potential properties they express at the time of interaction with specific clones of sensitive T lymphocytes, they are considered fundamentally important key players in immune reactions mediated by T lymphocytes. As a generalization imprecise but widely held, immature DCs are more "tolerogenic" than their more mature counterparts, while mature DCs are considered more "immunogenic" than their immature precursors. The ability of DCs, generated ex vivo from monocytes and armed with specific antigen, to function effectively in one of the two immunological directions, depends on their viability and vigor after being returned to the patient. It is logically concluded that the balance between competing immunostimulatory and immunosuppressive DCs will be an important determinant of both the direction and potency of DC-dependent therapeutic immune responses.

The purpose of the present invention is to facilitate the production of CD populations that lead in particular to the generation of powerful and clinically relevant immune responses. Although CD-based treatments, such as efforts to enhance cancer immunity, were very promising, overall clinical results have been disappointing. For example, Provenge, the first immunotherapy recently approved by the FDA for a solid tumor, adapting the conventional procedure of ex vivo production of CDs from blood monocytes, has only provided a four-month improvement in survival for patients with advanced prostate cancer. This conventional procedure of inducing DC, as well as extracorporeal photopheresis (ECF), an FDA-approved treatment for "liquid" malignancy cutaneous T-cell lymphoma (CTCL), is limited by the production of relatively heterogeneous CD populations. under conditions that impair the vigor and in vivo viability of the resulting DCs. By employing more physiological conditions for the production of therapeutic DCs, the present invention allows the production of more maturationally synchronized DCs, the survival and vigor of which are not inhibited by factors inherent in the process by which they are produced. Furthermore, this procedure is applicable to both human and animal leukocytes. DCs activate the responses of both CD8 + cytotoxic T lymphocytes (CTL) and CD4 + helper T lymphocytes (Th1). DCs can capture and process antigens and migrate to regional lymph nodes to present captured antigens and induce T cell responses. In humans, DCs are a relatively rare component of peripheral blood (<1% of leukocytes). However, laboratory procedures can differentiate large numbers of CDs from CD34 + precursors or blood monocytes.

For the properties described above, DCs have been identified as important cellular agents for eliciting effective antitumor immune responses. The idea is to generate DCs that present tumor-specific antigens in their MHC class I and MHC class II complex and can be (re) introduced into a patient to, in this way, launch an immune attack against the tumor. However, the generation of such immunostimulatory DCs usually requires differentiation of CD34 + precursors or blood monocytes using complex and rather expensive cytokine cocktails. In these standard procedures, cytokines are used at concentrations much higher (often by orders of magnitude) than those found in vivo under physiological conditions. Therefore, a proposed reason to justify the generally disappointing clinical results of DC-based immunomodulation is that DCs produced by the common cytokine procedure may not function effectively at the much lower cytokine concentrations in patients. DCs produced ex vivo at markedly higher than physiological concentrations of growth factors, such as cytokines, are selected to depend on conditions that reproduce in the in vivo environment of patients.

Classic extracorporeal photopheresis (ECF) has been used successfully to treat cutaneous T-cell lymphoma (CTCL) in subsets of patients. In ECF, patients with LCLT receive the photoactivatable compound 8-methoxypsoralen (8-MOP). Patients are then subjected to leukapheresis to obtain buffy coats and these buffy coats are passed through a continuous closed-loop ultraviolet exposure device to irradiate the buffy coats from the leukapheresis, thereby fatally damaging the lymphocytes. exposed. In this way, 8-MOP is induced to covalently crosslink DNA base pairs. The concept of ECF is to destroy proliferative metastatic T lymphocytes of the LCLT and then reintroduce via intravenous dying cells into the patient. It has been discovered that this procedure additionally results in the conversion of passed blood monocytes to CD without the need for stimulation by the addition of exogenous cytokines. Furthermore, these ECF-induced DCs are believed to internalize, process, and present antigens from tumor cells, which were killed by the combination of 8-MOP and UV radiation. It has been hypothesized that the reintroduction of these charged dendritic cells into the patient is responsible for at least part of the success of ECF in treating CTCL. In fact, procedures similar to ECF, in which neither 8-MOP nor UV light radiation is used, but in which the extracorporeal blood sample comprising monocytes is passed under shear stress through a FEC, are also believed to initiate the differentiation of monocytes into CDs.

However, the ECF or ECF-like procedure has also been found to result in truncated DCs, i.e., immunosuppressive or tolerogenic, which probably contributes greatly to the clinical efficacy of ECF in treating the disease. graft versus host disease, which commonly occurs after allogeneic stem cell / bone marrow transplants. The precise mechanistic aspects of ECF on the differentiation of monocytes into immunostimulatory or immunosuppressive DCs have yet to be finalized (for a review of the ECF procedure, see Girardi et al. (2002), Transfusion and Apheresis Science, 26, 181-190 ).

WO 2011/137365 describes methods for treating blood monocytes to produce functional antigen-presenting dendritic cells. An extracorporeal amount of blood from a subject is treated with a photoactivatable agent and passed through a plastic treatment device that allows light transmittance into the plastic device. A light source then produces light of a selected wavelength to activate the photoactivatable agent.

Berger et al. (2010, Blood, 116, 4838-4847) describe that ECF induces a high percentage of processed monocytes to enter the differentiation pathway of antigen-presenting dendritic cells in a single day, as determined by the enhanced expression of different genes.

Hoffmann et al. (2007, Journal of Investigative Dermatology, S117) describe the induction of immunomodulatory dendritic cells by transimmunization.

Engberg et al. (2007, Journal of Investigative Dermatology, S117) refer to the generation of tumor-specific anti-melanoma T lymphocyte responses by dendritic cells induced by extracorporeal photochemotherapy. Current FEC and FEC-like procedures are therefore designed to result in complex mixtures of immunostimulatory and immunosuppressive DCs. Of course, from a clinical perspective among others, it would be important to understand how ECF and ECF-like procedures can be modified to overcome these limitations and how immunostimulatory DCs can be obtained intentionally and selectively preferentially over immunosuppressive DCs and vice versa. Furthermore, the classical FEC procedure is, in principle, an in vivo procedure since the dendritic cell mixtures obtained are reinfused into the patient. However, it would be desirable to have procedures that allow the preferential production of immunostimulatory CDs over immunosuppressants and vice versa outside the human or animal body.

Thus, there is a continuing need for methods that allow for the predictable and reproducible production of individual-specific autologous immunostimulating dendritic cells, which can then be loaded with disease-specific antigens and which, after reintroduction, allow treatment of, for example , hyperproliferative diseases such as cancer.

OBJECTIVES AND SUMMARY OF THE INVENTION

An object of the present invention is to provide methods for producing immunostimulatory autologous dendritic cells.

Another object of the present invention is to provide methods for producing antigen-presenting autologous immunostimulating cells, preferably immunostimulating autologous dendritic cells.

Another object of the present invention is to provide methods for producing immunostimulating autologous dendritic cells in an extracorporeal amount of blood obtained from a patient.

A further objective of the present invention is to provide methods for producing immunostimulating autologous dendritic cells in an extracorporeal amount of blood obtained from a patient without the need for cytokine cocktails.

Still another object of the present invention is to provide methods for producing immunostimulating autologous dendritic cells preferentially in an extracorporeal amount of blood obtained from an individual such as a patient against immunosuppressive dendritic cells.

These and other objectives, as will be apparent from the following description hereinafter, are solved by the subject matter of the independent claims. Some of the preferred embodiments of the present invention form the subject matter of the dependent claims. Still other embodiments of the present invention can be taken from the following description.

The present invention is based to some extent on the data presented below in this document, which for a miniaturized and scalable device allowed (i) to imitate some aspects of the classical FEC procedure, (ii) to elucidate the cellular, molecular mechanism and the Biophysical conditions of induction of the differentiation of monocytes in immunostimulatory autologous dendritic cells in an extracorporeal quantity of blood. These data show that the activation of platelets and the binding of monocytes to said activated platelets under shear stress conditions is essential to obtain immunostimulatory autologous dendritic cells. As shown in the experiments described hereinafter, these immunostimulatory autologous dendritic cells can be characterized by the expression of molecular markers indicative of immunostimulating autologous dendritic cells. The data also show that conditions that result in increased glucocorticoid-induced leucine zipper (GILZ) expression will favorably allow monocytes to differentiate into immunosuppressive autologous dendritic cells. Thus, these findings allow a streamlined approach to obtaining immunostimulatory autologous dendritic cells by thus carefully selecting the properties of the devices to be used and the parameters of the procedure. The findings of the present invention make it possible to preferentially produce immunostimulatory dendritic cells over immunosuppressive dendritic cells and therefore overcome the limitations of the classic ECF procedure because, in the classical ECF procedure, the lack of knowledge of what type of dendritic cells are produced and how they are produced. Production can be manipulated to some extent prevents the expansion of the use of this procedure for applications other than those authorized (see Girardi et al. (2002), Transfusion and Apheresis Science, 26, 181-190). Furthermore, apart from for the classical ECF procedure as used in the device obtainable from Therakos, the present invention allows to obtain said immunostimulating dendritic cells in an experimental setting, where the extracorporeal amount of blood is not in continuous connection with the organism. The data suggest, inter alia, that the procedure for obtaining immunostimulating dendritic cells appears to include a step of global activation of monocytes and a subsequent step of differentiation of monocytes into immunostimulatory antigen-presenting cells (eg, dendritic cells). These steps appear to initially depend on the physical activation of monocytes with physical forces that occur during, for example, the initial purification or enrichment of monocytes that are sufficient for activation, although the passage of, for example, monocytes initially activated via devices as described herein may allow for enhanced activation and differentiation. Furthermore, if activation and differentiation take place in the absence of photoactivatable agents and UV-A (as used and has been used in FEC procedures), the formation of immunosuppressive dendritic cells appears to be favorably reduced as expression is reduced. by GILZ. Current data shed more light on the nature of molecular markers that can be used to identify immunostimulatory dendritic cells.

Some of the embodiments, which are based on this data, are described in more detail hereinafter. The present invention relates to a method for inducing the differentiation of monocytes contained in an extracorporeal quantity of a blood sample from a mammalian subject into immunostimulating autologous dendritic cells, said method comprising at least the steps of:

a) subjecting said extracorporeal quantity of said blood sample from a mammalian subject to a shear stress passing through a flow chamber that allows a fixed or adjustable flow rate adjustment;

b) wherein the procedure is performed in the absence of photoactivatable agents such as 8-MOP and UVA

c) wherein the procedure is performed without the need to add a molecular cocktail comprising cytokines; and

d) wherein the differentiation of monocytes from said blood sample into immunostimulatory autologous dendritic cells is identified by increased expression of at least one molecular marker HLA-DR, CD83, CD86, ICAM or PLAUR and non-increased expression of GILZ when comparing the immunostimulatory autologous dendritic cells with monocytes within the extracorporeal amount of a blood sample from a mammalian subject.

Additional suitable molecular markers are described herein below and can be taken, for example, from Table 1. These molecular markers can be grouped according to their known function as, for example, antigen presenting cell molecular markers, molecular markers cell adhesion, etc. Molecular markers whose expression is considered indicative of immunostimulatory dendritic cells include PLAUR, NEU1, CD80, CCR7, LOX1, CD83, ADAM-DEC, FPRL2, GPNMB, ICAM-1, HLA-DR and / or CD86. Markers such as HLA-DR, PLAUR and ICAM-1 can be considered indicative of global monocyte activation, whereas increased expression of, for example, CD83 and ADAM-DEC appears to be indicative of monocyte differentiation in cells. dendritic.

Monocyte activation is achieved among others, because said extracorporeal amount of said mammalian subject blood sample is subjected to a physical force by passing or cycling said extracorporeal amount of said mammalian subject blood sample through a flow chamber of a device, which allows adjusting the flow rate of said extracorporeal quantity of said mammalian blood sample through said flow chamber of said device so that a shear voltage is applied to said monocytes contained within said blood sample of mammalian subject.

Therefore, the activation of monocytes and the induction of differentiation into immunostimulating autologous dendritic cells can be achieved and influenced by varying the flow forces of the extracorporeal amount of the blood sample from the mammalian subject through the flow chamber of said device. , varying the geometry of the flow path of the flow chamber, varying the dimensions of the flow chamber, varying the temperature of the flow chamber and, therefore, the extracorporeal amount of the blood sample from a mammalian subject, changing the biophysical and geometric properties of the flow path surface etc.

As shown hereinafter, the activation of monocytes and the induction of differentiation into immunostimulating autologous dendritic cells can be optimized depending on the interaction of monocytes with activated platelets and / or specific plasma components in a situation where monocytes experience a physical force that can be provided by a device as described hereinafter.

The present disclosure therefore describes an activation of monocytes, which experience physical force and which interact with activated platelets and / or plasma components such as fibrinogen or fibronectin. Activation may be a procedure of subsequent steps that include the steps of (i) immobilizing plasma components such as fibrinogen or fibronectin as isolated components or as part of the extracorporeal amount of the blood sample from the mammalian subject in the flow chamber of said device (ii) passing platelets, which can be obtained as a purified fraction of the extracorporeal amount from the mammalian subject blood sample or as part of the extracorporeal amount from the mammalian subject blood sample, through the flow chamber so that platelets can interact with and be activated by plasma components and (iii) pass monocytes, which can be obtained as a purified fraction of the extracorporeal amount of the blood sample from the mammalian subject or as part of the extracorporeal amount of the blood sample from mammalian subject, through the flow chamber so that monocytes can interact with and activated by activated platelets and / or plasma components.

Therefore, in addition and / or alternatively to the parameters described above and to the variable adaptation of the architecture and the conditions under which the device is operated, the activation of monocytes and the induction of differentiation into immunostimulating autologous dendritic cells can be achieve and influence by varying the nature, purity and concentrations of the plasma components, the nature, purity and concentration of platelets, the order of the stages through which the plasma components and / or platelets are passed and / or are arranged in the flow chamber, the density with which the flow chamber is coated with the plasma components and / or platelets, the flow forces with which the extracorporeal amount of the blood sample of the mammalian subject is passed and , in particular, the platelets and / or monocytes through the flow chamber of said device, the temperature and / or the time in which the extracorporeal amount of mu The blood layer of a mammalian subject and, in particular, platelets and / or monocytes pass through the flow chamber of said device, etc.

However, it should be understood that, while such devices may be effective in particular in inducing activation and differentiation of monocytes into dendritic cells, the physical forces that monocytes experience during initial purification or enrichment, such as during Ficoll enrichment -Hypaque as described hereinafter, may already be sufficient to activate monocytes and induce their differentiation into immunostimulatory antigen presenting cells, such as dendritic cells. Similarly, although activated platelets and / or specific plasma components may be useful to increase the activation and differentiation of monocytes into immunostimulating dendritic cells, they may not be absolutely necessary. In order to effect the activation and differentiation of monocytes into immunostimulating dendritic cells, the invention therefore contemplates as a minimum requirement the application of physical forces. To allow this procedure to be carried out with the least possible influences, the invention always considers not applying molecular cocktails to achieve the maturation and differentiation of monocytes into immunostimulating autologous dendritic cells and to avoid conditions that give rise, for example, to increased expression of GILZ such as co-application of photoactivatable agents and UV-A.

In the above and below aspects and embodiment, the extracorporeal amount of the blood sample from the mammalian subject and, in particular, the monocytes, therefore, may or may not be obtained by apheresis such as leukapheresis.

Additionally, the invention also relates to said procedures that are carried out under conditions that avoid an increased expression of GILZ and / or an increased number of CD4 + CD25 + Foxp3 + lymphocytes and / or a down-regulation of CD80, CD86 and CD83. Thus, the invention relates to processes, which are carried out in the absence of a photoactivatable agent such as 8-MOP and without exposure to light such as UV-A.

The present disclosure also relates to methods as described hereinafter for obtaining autologous immunostimulatory antigen presenting cells, which can be used, for example, in immunization against tumor antigens, viral antigens, bacterial antigens or fungal antigens.

Another embodiment relates to methods as described hereinafter for producing immunostimulating dendritic cells which may be mammalian cells. Although the invention relates to the production of human immunostimulatory allogeneic dendritic cells, the present invention also contemplates using the methods for producing immunostimulatory dendritic cells from animals such as mice, rats, etc. These embodiments of the invention provide useful animal models and thus scalability of the methods and results described herein, eg, from mice to humans. Furthermore, since genetically identical lines of animals, such as mice, are available, immunostimulating dendritic cells from animals, such as immunostimulating dendritic cells from mice, can be introduced into the individual from whom the extracorporeal amount of blood sample was taken, and thus , be autologous in the strict sense or can be introduced into a genetically identical individual. This will, for example, allow any unexpected effects of these cells to be tested.

It should be understood that the methods described herein below have been shown to produce immunostimulating cells, which due to their molecular markers appear to be related to, if not corresponding to, cells that are commonly referred to as immunostimulating dendritic cells. Therefore, the immunostimulating cells according to the invention have been called immunostimulating dendritic cells.

The present disclosure also describes autologous immunostimulating dendritic cells or autologous immunostimulating antigen presenting cells obtainable by a method as described hereinbelow for use in immunization against tumor antigens, viral antigens, bacterial antigens, or fungal antigens.

Other embodiments will be described hereinafter.

LEGENDS OF THE FIGURES

Figure 1 Effect of platelet density on the number of monocyte / platelet interactions and subsequent monocyte phenotype. Monocytes were passed through parallel plates coated with platelets at low, medium or high density. (A) The number of monocyte / platelet interactions was substantially increased in plates coated with higher platelet densities. (B) After overnight incubation, monocytes that were exposed to high levels of platelets were significantly more likely to develop a phenotype consistent with DC differentiation, as assessed by expression of CD83 and HLA-DR in the membrane (high vs. medium or low density: p <0.0001; medium vs. low density: p <0.005). The data shown are the means (+/- SD) of at least 6 independent experiments. lpf: low magnification field.

Figure 2 Gene expression after exposure to platelets. Monocytes were exposed to high or low levels of platelets in flux. After overnight incubation, cells were evaluated for differences in gene expression using RT-PCR. Figure 2 shows changes in gene expression in monocytes exposed to high levels of platelets relative to those exposed to low levels. Seven genes associated with DC differentiation and / or function were found to be upregulated, while three were downregulated. Of the down-regulated genes, GPNMB and FPRL2 have known roles to reduce cytokine production and inhibit CD maturation, respectively. Of the upregulated genes, all have pro-immune functions or diverse roles in DC biology. See text for specific description of genes. The data shown are the means (+/- SD) of 2 independent experiments.

Figure 3 Influence of platelets on the differentiation of monocytes under static conditions. Monocytes were co-cultured for 18 hours with low, medium or high platelet concentrations under static conditions without flow. Under these conditions, no influence of platelets on DC differentiation was observed; all conditions resulted in low basal levels of cells expressing CD markers. Furthermore, activation of platelets with thrombin in the culture (blue line) did not cause an appreciable difference in the differentiation of monocytes relative to cultures containing platelets not activated by thrombin (red line).

Figure 4 Influence of plasma proteins on the adhesion of platelets to plates. Platelets were passed through plates coated with fibrinogen, plasma, fibronectin, or RMPI at the level of shear stress indicated on the x-axis. Platelets in flux were optimally adhered to fibronectin. For all proteins, platelet adhesion was maximum between 0.5 and 1.0 dynes / cm2lpf: low power field. The data shown are the means (+/- SD) of at least 2 independent experiments.

Figure 5 Influence of plasma proteins on the adhesion of platelets to plates coated with fibrinogen (A) or fibronectin (B). Platelets were either untreated (reference value) or pretreated with RGD fragments (+ RGD) or gamma fragments (+ Gamma) and their subsequent adhesion to fibrinogen (left panel) and fibronectin (right panel) was evaluated. Platelet binding to fibrinogen decreased with gamma fragments (p <0.05), while fibronectin binding decreased with RGD peptides (p <0.001). lpf: low magnification field. The data shown are the means (+/- SD) of at least 2 independent experiments.

Figure 6 Proteins involved in monocyte / platelet interactions. Monocytes were passed between platelet-coated plates with a wall shear stress of 0.5 dynes / cm2 under the conditions indicated on the x-axis: platelets pretreated with anti-P-selectin (P-) antibodies or with a control isotype (P +); monocytes pretreated with RGD peptides (RGD-) or with a control fragment (RGD +). Monocyte / platelet interactions were quantified under each set of conditions using digital microscopy, and are expressed in the figure as a fraction of the maximum observed under P + / RGD + conditions. Interactions were divided into those lasting less than 3 seconds (short duration, black bars) and those lasting more than 3 seconds, including stable binding (long duration, gray bars). All conditions that involved blocking with anti-P-selectin (P-) antibodies resulted in a significant decrease in interactions of short and long duration (**, p <0.01); blocking with RGD alone (RGD-) resulted in a significant decrease in interactions of long duration (*, p <0.05) but did not change interactions of short duration. The data shown are the means (+/- SD) of 3 independent experiments.

Figure 7 Effect of exposure to p-selectin on monocyte integrins. Plastic plates were coated with platelets at the specific gravity indicated on the x-axis. Platelets were then pretreated with anti-selectin antibodies (dashed line) or with an isotype control (gray line), or received no pretreatment (black line). Monocytes were passed through the plates at 0.5 dynes / cm2 and then immediately evaluated by flow cytometry for the expression of active p1 integrins. The y-axis indicates the percentage of monocytes that bind to an antibody directed against an epitope that is only exposed when the integrin is in the open conformation. The data shown are the means (+/- SD) of 3 independent experiments.

Figure 8 Effect of P-selectin exposure on monocyte phenotype after overnight incubation. Platelet coated plates were either untreated (first column) or pretreated with an isotype control (second column) or with anti-P-selectin antibodies (third column). Monocytes were passed through the plates at 0.5 dynes / cm2 and then incubated overnight. The y-axis indicates the percentage of monocytes that developed a phenotype consistent with the differentiation of DCs, that is, HLA-DR + / CD83 + in the membrane. The data shown are the means (+/- SD) of 3 independent experiments.

Figure 9 Proposed mechanism for induction of monocyte differentiation in CD. Based on the data presented in this manuscript, the following sequence of events is postulated: (1) plasma fibrinogen coats the plastic surface of the flow chamber; (2) through its receptor aIIbp3, non-activated platelets bind to the gamma component of immobilized fibrinogen; (3) platelets are activated and instantly express preformed P-selectin and other surface proteins; (4) Passed monocytes bind transiently to P-selectin via PSGL-1, causing partial activation of monocytes and conformational changes of the integrin receptor; (5) partially activated monocytes, which can now develop other interactions, bind to additional ligands expressed on platelets, including those containing RGD domains; (6) finally, influenced in this way, monocytes efficiently enter the CD maturation pathway within 18 hours. Note that, in vivo, step (1) above can be physiologically replaced by inflammatory signals from the tissue acting on the local endothelium, causing platelet recruitment and activation in a similar way.

Figure 10: GILZ expression is rapidly down-regulated as monocytes differentiate into immature CDMo, and are up-regulated after exposure to dexamethasone. A.) The expression of GILZ mRNA in CDMo CD11c + is presented as change in fold relative to freshly isolated monocytes. B.) Median fluorescence intensity for intracellular and cell surface markers after 0 and 36 h. C.) The expression of GILZ mRNA in CDMo CD11c + after 24 h is presented as change in fold relative to CDMo without dexamethasone. D.) The expression of GILZ mRNA in CDMo CD11c + is presented as change in times relative to CDMo at time 0 h. E.) Expression of GILZ mRNA in CD11c + CDMo after 24 hr as change in fold relative to untreated CDMo. F.) The expression of GILZ mRNA in CDMo CD11c + is presented as change in fold relative to untreated CDMo. All data are expressed as mean ± standard deviation for a minimum of 3 independent experiments. For differential gene expression: *> 2.5 times change and p <0.05, **> 2.5 times change and p <0.01, ***> 2.5 times change and p <0.001

Figure 11: 8-MOP plus UVA light up-regulate GILZ in immature CDMo in a dose-dependent manner. A.) The expression of GILZ is presented as a function of the concentration of 8-MOP at 1 J / cm2 and 2 J / cm2 of uVa light. GILZ mRNA expression in CDMo CD11c + 24 h after PUVA treatment is reported as fold change relative to CDMo without 8-MOP. B.) The expression of GILZ is presented as a function of the concentration of 8-MOP multiplied by the dose of UVA. C.) Percentage of early apoptotic CD11c + cells after 24 h. D.) Percentage of late apoptotic CD11c + cells after 24 h E.) Plots of cells selected by CD11c + are shown for UVA doses of 1 J / cm2 and 2 J / cm2 for 1 representative experiment of 4. The Percentage of CD11c + cells showing annexin-V + / 7-AAD- or annexin-V + / 7-AAD + phenotypes. The percentage of F.) CD11c + cells and G.) CD3 + cells expressing early and late apoptotic markers was quantified 24 h after treatment with 8-MOP (100 ng / ml) and UVA light (1 J / cm2). All data represent the mean ± standard deviation of at least 4 independent experiments. For differential gene expression: *> 2.5 times change and p <0.05, **> 2.5 times change and p <0.01

Figure 12: 8-MOP plus UVA light down-regulate CD83, CD80 and CD86 and up-regulate HLA-DR in immature CDMo in a dose-dependent manner. Relative fluorescence intensities for membrane expression of A.) HLA-DR and CD83, and B.) CD80 and CD86 are presented as a function of 8-MOP concentration (0 to 200 ng / ml) multiplied by the UVA dose (1 or 2 J / cm2) 24 h after PUVA treatment. The untreated MoDCs served as controls and were assigned an IFR value of 1. Data represent the mean ± standard deviation of 4 independent experiments. * p <0.05, ** p <0.01

Figure 13: Immature MoDCs exposed to apoptotic lymphocytes upregulate GILZ. A.) GILZ mRNA expression in CD11c + CDMo 24 h after coculture is reported as fold change relative to untreated CDMo that were cultured alone. B.) GILZ mRNA expression in CD11c + CDMo 24 h after coculture is presented as fold change relative to untreated CDMo that were cultured alone. C.) Relative fluorescence intensity for intracellular GILZ 24 h after coculture. The relative fluorescence intensities after and before LPS stimulation for D.) CD80 and CD86 and E.) HLA-DR and CD83 were calculated as follows: (IFM treated after LPS - IFM treated before LPS) / (IFM not treated after LPS - IFM not treated before LPS). Data represent the mean ± standard deviation of at least 4 independent experiments. For differential gene expression: *> 2.5-fold change and p <0.05 Figure 14: GILZ-expressing CDMo increase IL-10 production and decrease the production of various pro-inflammatory cytokines and chemokines. After 24 h of LPS stimulation, culture supernatants were collected for cytokine quantification by multiple immunoassays with magnetic microspheres for A.) IL-10 and pro-inflammatory cytokines B.) IL-12p70 and ^IFN- y, ^{C .) IL-6 and TNF-a. The same analysis was performed for the pro-inflammatory chemokines D.) IL-8 and} E.) MCP-1, MIP-1 p and RANTES. Data are presented as mean ± standard deviation of 3 independent experiments. * p <0.05 compared to the untreated CDMo group.

Figure 15: SiRNA-mediated attenuation of GILZ neutralizes the increased ratio between IL-10 and IL-12p70 characteristic of tolerogenic DCs. A.) GILZ mRNA expression is presented as fold change compared to untreated CDMo that were cultured alone. *> 2.5 times change and p <0.05. B.) Quantification of the levels of IL-10 and IL-12p70 proteins in culture supernatants after stimulation with LPS. Data represent the mean ± standard deviation of 3 independent experiments. * p <0.05, compared to identically treated CDMo not transfected with siRNA.

Figure 16: Represents the flow of monocytes in a classic FEC procedure in the presence of UVA and 8-MOP.

Monocytes in the middle experience less exposure to UVA rays than monocytes towards the channel surfaces.

Figure 17: Represents the layout of the device channels used in a classic FEC procedure.

Figure 18: a) to d) represent different geometries of the flow chamber of a device that can be used for the methods of the invention.

Figure 19: A) represents the geometry of a device used in some of the examples. B) represents the geometry of an alternative device.

Figure 20: Represents the increase in HLA-DR expression after physical activation of monocytes through a device of figure 19

Figure 21: Represents the increase in complexity by FSC / SSC after physical activation of monocytes through a device of figure 19

Figure 22: Represents the increase in complexity by FSC / SSC after physical activation of monocytes when passing through a device of figure 19

Figure 23: Represents the increase in the expression of HLA-DR, CD86, ICAM-1, PLAUR and / or the complexity by FSC / SSC after the physical activation of monocytes through a device of figure 19 DETAILED DESCRIPTION OF THE INVENTION

Before describing the invention in detail with respect to some of its preferred embodiments, the following general definitions are provided.

The present invention as illustratively described in the following may be suitably practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein.

The present invention will be described below with respect to particular embodiments and with reference to certain figures, but the invention is not limited by them, but only by the claims.

When the term "comprising" is used in the present description and in the claims, it does not exclude other elements. For the purposes of the present invention, the term "consisting of" is considered to be a preferred embodiment of the term "comprising". If hereinafter a group is defined as comprising at least a certain number of embodiments, this should also be understood as disclosing a group that preferably consists only of these embodiments.

For the purposes of the present invention, the term "obtained" is considered to be a preferred embodiment of the term "obtainable". If hereinafter, for example, an antibody is defined as obtainable from a specific source, this should also be understood as disclosing an antibody obtained from this source.

When an indefinite or definite article is used in reference to a singular noun, for example, "a", "an" or "the", this includes a plural of that noun unless specifically stated otherwise. In the context of the present invention, the terms "about" or "about" indicate a range of accuracy that one skilled in the art will understand to still guarantee the technical effect of the characteristic feature in question. The term typically indicates deviation from the indicated numerical value of ± 20%, preferably ± 15%, more preferably ± 10%, and even more preferably ± 5%.

Furthermore, the terms "first", "second", "third" or "(a)", "(b)", "(c)", "(d)" or "(i)", "(ii) "," (iii) "," (iv) ", etc. and the like in the description and in the claims, are used to distinguish between similar items and not necessarily to describe a sequential or chronological order. It should be understood that terms used in this way are interchangeable under appropriate circumstances and that the embodiments of the invention described herein may function in sequences other than those described or illustrated herein.

In case the terms "first", "second", "third" or "(a)", "(b)", "(c)", "(d)" or "(i)", "( ii) "," (iii) "," (iv) "etc. refer to steps in a procedure or use or assay, there is no chronological or time interval consistency between steps unless otherwise indicated, i.e. steps may be carried out simultaneously or there may be time gaps seconds, minutes, hours, days, weeks, months or even years between such stages, unless otherwise indicated in the application as set forth above or below. Technical terms are used in the usual sense. If a specific meaning is expressed by certain terms, the definitions of the terms will be given as follows in the context in which the terms are used.

As already mentioned, the present invention is based to some extent on the data presented below in the present document, which for a miniaturized device allowed (i) to imitate some aspects of the classical FEC procedure, and (ii) to elucidate the mechanism Cellular and molecular induction of monocyte differentiation into immunostimulatory dendritic cells in an extracorporeal amount of blood. How I know shown in the experiments described herein below, these immunostimulatory autologous dendritic cells can be characterized by the expression of molecular markers indicative of immunostimulatory autologous dendritic cells. The data also show that conditions that result in increased glucocorticoid-induced leucine zipper (GILZ) expression will favorably allow monocytes to differentiate into immunosuppressive autologous dendritic cells. For the purposes of the present invention, such immunosuppressive autologous dendritic cells are also referred to as immunoinhibitory autologous dendritic cells, tolerogenic autologous dendritic cells or truncated autologous dendritic cells. These data show that the sequential activation of platelets and the binding of monocytes to said activated platelets under shear stress conditions is essential to obtain immunostimulating dendritic cells. Furthermore, these findings immediately allow for a streamlined approach to obtaining immunostimulatory dendritic cells. Since the series of molecular events that give rise to the formation of immunostimulating autologous dendritic cells and immunosuppressive autologous dendritic cells obtained in the classical procedure of ECF can be mimicked and dissected, devices and more particularly flow chambers can now be designed, which allow further dissection of the molecular events that give rise to the differentiation of monocytes into immunostimulating autologous dendritic cells on a scale suitable for research purposes, but which also allow obtaining said immunostimulatory autologous dendritic cells for therapeutic purposes. This will be explained in more detail.

In the classical FEC procedure, typically 2.5 to 6 L of blood is obtained from patients suffering from CTCL by apheresis, such as leukapheresis. This extracorporeal amount of blood, which is typically processed by apheresis, such as leukapheresis to give a final volume of approximately 200 ml to 500 ml comprising leukocytes including monocytes, as well as plasma components, platelets and cancerous T lymphocytes, is then passed under shear stress through a photopheresis device having transparent plastic channels in conjunction with the photoactivatable drug 8-MOP. This extracorporeal amount of blood comprising 8-MOP is then radiated by exposing the transparent channels to UV-A having a wavelength of 315 to 380 nm. The extracorporeal amount of radiated blood is then reintroduced into the patient. The beneficial effects of this procedure on the evolution of CTCL in some of the treated patients were initially assumed to be the result of the destruction of cancerous T cells. Based on this hypothesis, it was assumed that patients would have to undergo repeated cycles of ECF. However, in some of the patients, long-term beneficial effects were observed, making repeated treatment superfluous and subsequently interesting and partially unreconcilable effects were found, which could explain some of the positive results of ECF for treatment of the LCCT.

For example, as described in US 6,524,855, CD induction was observed in the extracorporeal amount of blood and it was hypothesized that some of the beneficial effects of ECF on CTCL were due to cancer-specific antigens. that cancerous T lymphocytes were released into the circulation as a consequence of the 8-MOP-induced apoptosis of these cells and the loading of DCs, which had begun to differentiate, with these antigens. The reintroduction of the extracorporeal amount of blood comprising such autologous DCs loaded with cancer antigen was assumed to provide a long-term durable therapeutic effect similar to vaccination. However, at the same time it was observed that during the ECF procedure so-called "truncated" DCs were formed, which did not provide an immunostimulating effect, but quite the opposite, namely an immunosuppressive effect. The induction of these different types of DCs with opposite effects by the same procedure was puzzling and, from a practical perspective, posed obstacles to the rationalized use of ECP to obtain immunostimulatory or immunosuppressive DCs. Furthermore, the need for apheresis, such as leukapheresis, to obtain a sufficient amount of extracorporeal blood is another factor that negatively affects the quality of treatment for patients.

The data presented hereinafter suggest that shear stress is in principle responsible for the overall activation of monocytes and induction of CD. By using, for example, the miniaturized model device as described hereinafter, it was shown that induction of immunostimulating DCs occurs even if substantially smaller amounts of extracorporeal blood are used, which have not been obtained by apheresis, such as leukapheresis, even if 8-MOP is not added to the extracorporeal amount of blood and even if UV-A radiation is not performed. Therefore, the induction of DCs occurred despite the omission of the main steps of the classical procedure of ECF. However, shear stress seems to be a fundamental factor in obtaining immunostimulating DCs. Other stages with a positive influence on the induction of DC formation appear to be the activation of platelets by plasma components and the activation of monocytes by these activated platelets. The data further suggest that, if shear stress-induced induction of DC formation occurs in the presence of 8-MOP and UVA radiation, glucocorticoid-induced leucine zipper (GILZ) expression is increased, thereby which in turn activates a pathway that leads to the formation of truncated tolerogenic DCs, that is, immunosuppressive (see example 2). The fact that the induction of immunostimulatory DCs induced by the shear voltage can be achieved by applying the shear voltage without the addition of 8-MOP and without UV-A radiation further suggests that in the classical procedure of FEC, due to the dimensions of the plastic channels, some of the DCs induced by shear stress were not initially radiated effectively with the consequence that these DCs could also become immunostimulatory DCs (see Figure 16). These above data were obtained using a device having the general architecture of Figure 17. However, in classical FEC and FEC-like procedures, obtained mixtures of immunostimulatory autologous and immunosuppressive autologous dendritic cells. Based on the data presented hereinafter, it is now possible, for example, to dispense with some of the requirements of the FEC procedures and similar to the prior art FEC, for example the use of large amounts of blood requiring apheresis processing, such as leukapheresis. Furthermore, one can now deliberately adapt the parameters of the procedure and the design of the device, which is used to exert a physical force on monocytes, to deliberately obtain immunostimulating autologous or immunosuppressive autologous dendritic cells.

The procedure as described hereinafter is performed without the need for molecular cocktails to achieve maturation and differentiation of monocytes into immunostimulatory autologous dendritic cells. Furthermore, as the invention is based on inducing the differentiation of monocytes contained in an extracorporeal amount of blood sample from a mammalian subject, the differentiation procedure is not limited to molecular events that can be triggered by typical cytokine cocktails. In contrast, dendritic cells obtainable with the procedures described herein below appear to have more complex, yet synchronized, molecular patterns that appear to be representative of the broader functionality of these dendritic cells.

The present invention relates to a method for inducing the differentiation of monocytes contained in an extracorporeal quantity of a blood sample from a mammalian subject into immunostimulating autologous dendritic cells, said method comprising at least the steps of:

a) subjecting said extracorporeal quantity of said blood sample from a mammalian subject to a shear stress passing through a flow chamber that allows a fixed or adjustable flow rate adjustment;

b) wherein the procedure is performed in the absence of photoactivatable agents such as 8-MOP and UVA c) wherein the procedure is performed without the need to add a molecular cocktail comprising cytokines; and

d) wherein the differentiation of monocytes from said blood sample into immunostimulatory autologous dendritic cells is identified by increased expression of at least one molecular marker HLA-DR, CD83, CD86, ICAM or PLAUR and non-increased expression of GILZ when comparing the immunostimulatory autologous dendritic cells with monocytes within the extracorporeal amount of a blood sample from a mammalian subject.

and)

It is to be understood that the invention, as well as all of the embodiments described herein below, can preferably be used to provide immunostimulating autologous dendritic cells, namely, if the obtained immunostimulating dendritic cells are later reintroduced into the same donor. This can be done continuously or discontinuously, where the dendritic cells are cultured for extended periods of time before being reintroduced into the donor. As this will be the preferred application, all of the embodiments discussed hereinafter relate to immunostimulatory autologous dendritic cells. However, it should be understood that the analysis of such embodiments always includes the scenario where the invention is used to prepare immunostimulatory dendritic cells as such and where only subsequent administration of these cells will convert them into potentially immunostimulatory autologous dendritic cells.

As already mentioned, the methods described herein below have been shown to produce immunostimulating and immunosuppressive cells which, due to their molecular markers, appear to be related to, if not corresponding to, cells commonly referred to as dendritic cells. Therefore, the immunostimulating cells according to the invention have been called immunostimulating dendritic cells.

The term "immunostimulatory autologous dendritic cells" therefore refers to cells that can be derived from monocytes by treating monocytes contained in an extracorporeal amount of said mammalian subject blood sample as described herein and which are identified by at least one of the molecular markers HLA-DR, CD83, CD86, ICAM or PLAUR and not increased expression of GILZ as described in the following. These molecular markers have been analyzed in the literature for dendritic cells that can present antigens via MHC I and MHC II. It should be understood that immunostimulating autologous dendritic cells obtainable by the methods described herein and identified by the molecular markers described herein can be considered dendritic cells that have already sufficiently differentiated and that have internalized and even present, for example, antigens. tumor-specific apoptotic cells such as cytotoxic T lymphocytes, which are contained in the extracorporeal amount of a respective mammalian subject blood sample, or for example, viral or bacterial antigens, which are contained in the extracorporeal amount of a blood sample mammalian subject respective, so that they can be considered or be immunostimulating autologous antigen-presenting dendritic cells. However, the procedure can also be carried out in such a way that the dendritic cells express molecular markers indicative of immunostimulating dendritic cells, which have not yet internalized and do not present antigens. It is necessary to understand that where dendritic cells are mentioned herein, it refers to immunostimulatory antigen presenting cells that have the ability to present, for example, disease-specific antigens on their surfaces after these cells have been contacted with said antigens.

The present invention enables the preferential production of immunostimulatory DCs over immunosuppressive DCs. The preferential production of immunostimulating dendritic cells over immunosuppressive dendritic cells means that starting from an extracorporeal amount of blood sample, more immunostimulating dendritic cells can be obtained than immunosuppressive DC compared to a situation where, for example, the same extracorporeal amount blood sample is subjected to a classical fEc procedure. Although the production of immunostimulating DCs will occur preferentially over the production of immunosuppressive DCs, there may still be immunosuppressive DCs after the procedures according to the invention have been performed. However, the present invention provides the parameters and variables that can be manipulated to favor the production of one dendritic cell population over the other. The production of immunostimulatory dendritic cells is achieved without using 8-MOP or UVA and by culturing the obtained immunostimulating dendritic cells for prolonged periods of time, such as 1, 2, 3, or 4 days. In this way, the formation of immunosuppressive dendritic cells can be reduced to a clinically acceptable level. Furthermore, the immunosuppressive dendritic cells that eventually remain can be eliminated, for example, by affinity purification against molecular markers that are indicative of immunosuppressive dendritic cells.

As described in the examples, molecular markers that are indicative of immunostimulating autologous dendritic cells obtainable by the procedures described herein were identified by subjecting monocytes contained in the extracorporeal amount of blood samples from mammalian subjects derived from healthy volunteers to the procedure using a miniaturized device (see markers 88 to 99 in Table 1). Furthermore, as is also described in the example, molecular markers that are indicative of immunostimulating autologous dendritic cells were identified, subjecting monocytes contained in the extracorporeal amount of blood samples from mammalian subjects derived from healthy volunteers or from patients suffering from LCLT or disease of ICH (GVHD) to a FEC procedure (see markers 1 to 87 of Table 1). The dendritic cells were then isolated and analyzed for upregulated expression of molecular markers, which are known or suspected to play a role in immunostimulating dendritic cells. Some of the markers identified for the FEC procedure, which are assumed to give rise to a complex mixture of immunostimulatory and immunosuppressive dendritic cells, are the same as those observed for dendritic cells obtained by the miniaturized device procedure. which should give rise only to immunostimulating dendritic cells. Therefore, to the extent that the ECF procedure leads to upregulation of molecular markers, which may be associated with dendritic cell function, it seems justified to assume that these markers will also be suitable for identifying obtainable immunostimulatory dendritic cells. by the procedures described herein, such as with the miniaturized device. A set of 99 molecular markers in total were identified as upregulated in immunostimulating autologous dendritic cells obtainable by the methods described herein. This set can be expanded with other molecular markers in the future through similar analyzes.

Thus, the data from Examples 1 and 3 yield a set of 99 markers, which are considered indicative of immunostimulatory autologous dendritic cells. These markers are summarized in Table 1.

Table 1

Of the 87 genes (markers 1 to 87 in Table 1) that represent surface markers / functional mediators of immunostimulatory DC function, 66 were found to be uniquely identified in dendritic cells induced by the ECF procedure ( passed through the plate, grown overnight, see example), after comparing with expression databases for "classic" dendritic cells. These are: ABCA1, ACVR1B, ATP6V0B, BASP1, BEST1, CPM, CRK, CSF2RA, CTNND1, CTSB, CXCL16, ENG, FLOT1, GNA15, GPR137B, GPR157, HEXB, HOMER3, ICAM1, IRAK11GB8, ITGA5, ITGA5CT , LEPROT, MARCKSL1, MCOLN1, MFAP3, MGAT4B, MR1, MRAS, MSR1, NEU1, OLR1, OMG, PI4K2A, PLAUR, PMP22, PVRL2, RAB13, RAB8B, RAB9A, RALA, RNASE1, SC5LCDL, SMA4, SPA6 , SLC31A1, SLC35E3, SLC39A6, SLC6A6, SLC6A8, STX3, STX6, TM9SF1, TMBIM1, TMEM33, TNFRSF10B, TNFRSF11A, TNFRSF1A, TNFRSF1B, TNFSF14, TNFSF9, YKT6.

Therefore, immunostimulatory autologous dendritic cells are identified by determining expression of at least one molecular marker of HLA-DR, CD83, CD86, ICAM or PLAUR and non-increased expression of GILZ for immunostimulatory autologous dendritic cells obtainable by the procedures described herein. document and comparing their expression for monocytes contained within the extracorporeal amount of a blood sample from a mammalian subject. If increased expression is observed in immunostimulatory autologous dendritic cells against monocytes, this is indicative of monocyte differentiation into immunostimulatory autologous dendritic cells.

In accordance with the present disclosure, immunostimulatory autologous dendritic cells can be identified by determining the expression of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more additional selectable molecular markers from Table 1. For example, immunostimulatory autologous dendritic cells can be identified by determining the expression of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 additional molecular markers that can be selected from the group comprising PLAUR, NEU1, CTSB, CXCL16, ICAM1, MSR1, OLR1, SIRPA, TNFRSF1A , TNFSF14, TNFSF9, PMB22, CD40, LAMP3, CD80, CCR7, LOX1, CD83, ADAM-DEC, FPRL2, GPNMB and / or CD86. More preferably, immunostimulatory autologous dendritic cells can be identified in accordance with the present disclosure by determining the expression of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 molecular markers that can be selected from the group comprising PLAUR, NEU1, CD80, CCR7, LOX1, CD83, ADAM-DEC, FPRL2, GPNMB and / or CD86. The most preferred markers, which are considered indicative of immunostimulatory autologous dendritic cells according to the present disclosure are PLAUR, NEU1, CD80, CD83 and / or CD86.

The data and conclusions presented herein suggest that the method of obtaining immunostimulating dendritic cells appears to include a global activation step of monocytes and a step of differentiation of monocytes into immunostimulating antigen-presenting cells (eg, dendritic cells). It appears that these different steps can be traced by molecular markers as described above and by forward scatter / side scatter complexity (FSC / SSC complexity) which can be determined by FACS analysis. In addition, molecular markers can be grouped according to their known function as, for example, antigen presentation molecular markers, cell adhesion molecular markers, etc. HLA-DR, CD86, and CD 80 can be considered representative of antigen presentation. PLAUR and ICAM-1 can be considered representative of cell adhesion. Markers such as HLA-DR, PLAUR and ICAM-1, as well as FSC / SSC complexity, can also be considered indicative of global activation of monocytes, while increased expression of, for example, CD83, ADAM-DEC, CD40, CD80, LAMP-3, and CCR7 seem indicative of monocyte differentiation into dendritic cells.

As described herein, if procedures are carried out to allow increased expression of GILZ (SEQ ID NO: 100), IDO (indolamine) (SEQ ID NO: 101), KMO (kynurenine 3-hydroxylase) ( SEQ ID NO: 102), transforming growth factor beta (TGFp) (SEQ ID NO: 103), and / or IL-10 (interleukin 10) (SEQ ID NO: 104), the monocytes contained within the extracorporeal amount of a blood sample from a mammalian subject will not differentiate into immunostimulating autologous dendritic cells, but into immature dendritic cells, termed truncated or immunosuppressive. Therefore, immunostimulating autologous dendritic cells are identified not only by determining the expression of the molecular markers mentioned above, but also by determining that GILZ expression is not increased in immunostimulating autologous dendritic cells against monocytes. If increased GILZ expression were determined, this would be considered indicative that at least some immunosuppressive dendritic cells have formed. Marker molecular that is currently considered indicative of immunosuppressive dendritic cells is GILZ. Markers that can be further considered are IDO, KMO, TGFp and / or IL10.

As mentioned above, the procedure as described hereinafter is performed without the need for molecular cocktails to achieve the maturation and differentiation of monocytes into immunostimulatory autologous dendritic cells. Such cocktails can comprise factors such as, for example, IL- ^{4, GM-CSF, LPS, IFN-} y, ^{IL-1 p and TNF-α.}

Therefore, immunostimulating autologous dendritic cells obtainable by the methods described herein can not only be positively identified by molecular markers that are indicative of immunostimulating dendritic cells such as PLAUR, CD80, and CD83, but also by the absence of upregulation. of molecular markers that are indicative of immunosuppressive dendritic cells, namely GILZ. Furthermore, these two types of cells, that is, immunostimulating and immunosuppressive dendritic cells, can be distinguished from monocytes, which are subjected to a shear stress to induce their differentiation process, determining the expression of molecular markers that they are considered indicative of monocytes such as CD33, CD36 and / or FCGR1a (receptor for fragment 1A of IgGFc). If the expression of these factors is found to be down-regulated compared to the expression of monocytes, before they have been subjected to a shear stress and procedure as described herein, then this is considered indicative that monocytes have entered the maturation and differentiation pathway to give immunostimulating and / or immunosuppressive dendritic cells. The distinction between these last two dendritic cell populations can then be made by determining the expression of molecular markers such as PLAUR, ICAM-1, CD80, CD83, and GILZ.

Since the knowledge and corresponding tools are now available, for example, the molecular markers available to distinguish between immunostimulating autologous dendritic cells and immunosuppressive autologous dendritic cells, it is now possible to deliberately vary both the device design and the flow chamber to through which the extracorporeal amount of a blood sample from a mammalian subject and, therefore, the monocytes, is passed so that they experience a shear stress, and the parameters with which the procedure for inducing monocyte differentiation is carried out in immunostimulatory autologous dendritic cells, to deliberately allow the differentiation of monocytes into immunostimulatory autologous dendritic cells.

As mentioned above, an extracorporeal amount of a blood sample from a mammalian subject is passed through a flow chamber of a device, so that a shear voltage is applied to said monocytes contained within said blood sample from mammalian subject. Flow chamber and device design alterations influencing monocyte differentiation into immunostimulatory autologous dendritic cells include variation of flow forces, variation of flow path geometry of the flow chamber, variation of dimensions of the flow chamber, the possibility of adjusting the temperature, the possibility of exposing the extracorporeal amount of the blood sample from the mammalian subject in the flow chamber to visible light, etc. The application of a shear stress can be achieved not only, for example, by passing an extracorporeal amount of blood sample through a flow chamber, but also by placing said extracorporeal amount of blood sample in, for example, a bag of EVA plastic available from Macopharma and gently shaking or shaking this bag filled with blood sample (see, for example, Andreu et al., (1994), Trans. Sci., 15 (4), 443-454)

As also mentioned above and shown hereinafter, the activation of monocytes and the induction of differentiation into immunostimulatory autologous dendritic cells is dependent on the interaction of monocytes with activated platelets and / or specific plasma components in a specific situation. wherein the monocytes experience a shear stress that can be provided by a device as described herein below. Variation of the process parameters therefore includes varying the nature, purity and concentrations of plasma components, the nature, purity and concentration of platelets, the order of stages through which plasma components and / or platelets pass and / or are dispose in the flow chamber, the density with which the flow chamber is coated with plasma components and / or platelets, the flow forces of the extracorporeal quantity of the blood sample from the mammalian subject and, in particular, the platelets and / or the monocytes that pass through the flow chamber of said device, the temperature and / or time in which the extracorporeal amount of the blood sample from the mammalian subject and, in particular, the platelets and / or the monocytes pass to through the flow chamber of said device, etc.

Factors related to device and flow chamber design, as well as procedural parameters, will now be discussed in more detail as to their relevance to the differentiation of monocytes into immunostimulatory autologous dendritic cells. It should be understood that in any of the embodiments discussed in the following, the differentiation of monocytes into immunostimulating autologous dendritic cells is achieved, wherein the immunostimulating autologous dendritic cells are identified by determining the expression of molecular markers described above and determining the expression of GILZ. . Furthermore, for all the embodiments discussed in the following, it should be understood that monocytes that are contained in an extracorporeal amount of a mammalian subject blood sample are subjected to shear stress to allow them to differentiate into immunostimulatory autologous dendritic cells, for example, upon interaction with activated platelets and / or plasma components.

The invention relates to a method for inducing the differentiation of monocytes contained in an extracorporeal amount of a blood sample from a mammalian subject into immunostimulating autologous dendritic cells, wherein according to claim 1, said extracorporeal amount of said blood sample of mammalian subject is subjected to a physical force by passing said extracorporeal amount of said mammalian subject blood sample through a flow chamber of a device, which allows adjusting the flow rate of said extracorporeal amount of said mammalian subject blood sample through said flow chamber of said device so that a shear stress is applied to said monocytes contained within said mammalian subject blood sample.

In one embodiment, the invention of claim 1 refers to a method for inducing the differentiation of monocytes contained in an extracorporeal amount of a blood sample from a mammalian subject into immunostimulating autologous dendritic cells, wherein said extracorporeal amount of said A sample of blood from a mammalian subject is subjected to a physical force by passing said extracorporeal amount of said blood sample from a mammalian subject through a flow chamber of a device, which allows adjusting the flow rate of said extracorporeal amount of said blood sample of mammalian subject through said flow chamber of said device such that a shear stress is applied to said monocytes contained within said mammalian subject blood sample, and wherein said flow chamber of said device has a design that allows applying a shear voltage to said monocytes contained within said blood sample of subject to mammal.

In another embodiment, the invention of claim 1 refers to a method for inducing the differentiation of monocytes contained in an extracorporeal amount of a blood sample from a mammalian subject into immunostimulating autologous dendritic cells, wherein said extracorporeal amount of said A sample of blood from a mammalian subject is subjected to a physical force by passing said extracorporeal amount of said blood sample from a mammalian subject through a flow chamber of a device, which allows adjusting the flow rate of said extracorporeal amount of said blood sample of mammalian subject through said flow chamber of said device such that a shear voltage is applied to said monocytes contained within said mammalian subject blood sample, and wherein said device additionally allows adjustment of at least one parameter selected from the group comprising temperature and light exposure.

In another embodiment, the invention of claim 1 refers to a method for inducing the differentiation of monocytes contained in an extracorporeal amount of a blood sample from a mammalian subject into immunostimulating autologous dendritic cells, wherein said extracorporeal amount of said blood sample from a mammalian subject is subjected to shear stress by passing said extracorporeal amount of said blood sample from a mammalian subject through a flow chamber of a device as mentioned above and in which said monocytes are activated and induced to differentiate into immunostimulating autologous dendritic cells through interaction with activated platelets and / or plasma components.

For example, in one embodiment, the invention of claim 1 relates to a method for inducing the differentiation of monocytes contained in an extracorporeal amount of a blood sample from a mammalian subject into immunostimulating autologous dendritic cells, wherein said method It comprises at least the stages of:

a) applying said extracorporeal amount of said mammalian subject blood sample comprising at least monocytes to a device, which is configured to provide a flow chamber through which said extracorporeal amount of said subject blood sample can be passed mammalian, b) activating platelets, which may be comprised within said extracorporeal amount of said mammalian subject blood or which may be provided separately from said mammalian subject blood sample comprising at least monocytes,

c) treating said extracorporeal amount of said mammalian subject blood sample comprising at least monocytes in said device by applying a physical force to the monocytes contained within said extracorporeal amount of said mammalian subject blood sample such that said monocytes are activated and are induced to differentiate into immunostimulatory autologous dendritic cells by binding to said activated platelets obtained in step b).

In another embodiment, the invention of claim 1 relates to a method for inducing the differentiation of monocytes contained in an extracorporeal amount of a blood sample from a mammalian subject into immunostimulating autologous dendritic cells, wherein said method comprises at least the stages of:

a) applying said extracorporeal amount of said mammalian subject blood sample comprising at least monocytes to a device, which is configured to provide a flow chamber through which said extracorporeal amount of said subject blood sample can be passed mammal, b) passing plasma components, which may be comprised within said extracorporeal amount of said mammalian subject blood sample or which may be provided separately from said mammalian subject blood sample,

c) treating said extracorporeal amount of said mammalian subject blood sample comprising at least monocytes in said device by applying a physical force to the monocytes contained within said extracorporeal amount of said mammalian subject blood sample such that said monocytes are activated and they are induced to differentiate into immunostimulating autologous dendritic cells by binding to said plasma components obtained in step b).

In still another embodiment, the invention of claim 1 relates to a method for inducing the differentiation of monocytes contained in an extracorporeal amount of a blood sample from a mammalian subject into immunostimulating autologous dendritic cells, wherein said method comprises at minus the stages of:

a) applying said extracorporeal amount of said mammalian subject blood sample comprising at least monocytes to a device, which is configured to provide a flow chamber through which said extracorporeal amount of said subject blood sample can be passed mammal, b) passing plasma components, which may be comprised within said extracorporeal amount of said mammalian subject blood or which may be provided separately from said mammalian subject blood sample,

c) activating platelets, which may be comprised within said extracorporeal amount of said mammalian subject blood sample or which may be provided separately from said mammalian subject blood sample comprising at least monocytes,

d) treating said extracorporeal amount of said mammalian subject blood comprising at least monocytes in said device by applying a physical force to the monocytes contained within said extracorporeal amount of said mammalian subject blood sample such that said monocytes are activated and activated. they induce differentiation into immunostimulatory autologous dendritic cells by binding to said activated platelets and / or plasma components obtained in steps b) and c).

In still another embodiment, the invention of claim 1 relates to a method for inducing the differentiation of monocytes contained in an extracorporeal amount of a blood sample from a mammalian subject into immunostimulating autologous dendritic cells, wherein said method comprises at minus the stages of:

a) optionally passing platelet rich plasma through a device that is configured to provide a flow chamber through which said extracorporeal amount of said mammalian subject blood sample can be passed,

b) applying said extracorporeal amount of said mammalian subject blood sample comprising at least monocytes to a device, which is configured to provide a flow chamber through which said extracorporeal amount of said subject blood sample can be passed mammal, c) treating said extracorporeal amount of said mammalian subject blood comprising at least monocytes in said device by applying physical force to the monocytes contained within said extracorporeal amount of said mammalian subject blood sample so that said monocytes are activated and are induced to differentiate into immunostimulatory autologous dendritic cells optionally by binding to said platelet-rich plasma of steps a).

As can be deduced from the experiment described herein, the procedure for inducing the differentiation of monocytes contained in an extracorporeal amount of blood from a mammalian subject into immunostimulating autologous dendritic cells works optimally if the platelets that are comprised within said extracorporeal amount of said mammalian subject blood are activated and if the extracorporeal amount of said mammalian subject blood comprising at least monocytes in said device is treated by applying a shear voltage to the monocytes contained within said extracorporeal amount of said mammalian subject blood of so that these monocytes are activated and induced to differentiate into immunostimulating autologous dendritic cells by binding to said activated platelets. However, monocyte activation can also be achieved by direct interaction with plasma components, that is, without interaction with activated platelets.

The steps to activate platelets and the subsequent activation and differentiation of monocytes in CD will be discussed in the following for the embodiment in which (i) plasma components, such as plasma proteins, are passed through the flow chamber of the device such that these components adhere to the walls of the flow chamber, in which (ii) platelets are passed through the flow chamber and activated by binding to plasma components and in which (iii) fractions containing Monocytes, such as an extracorporeal amount of said mammalian subject blood comprising at least monocytes are passed through the flow chamber and activated to differentiate into CDs by binding to activated platelets. However, it should be understood that these activities also occur if the plasma fraction or plasma proteins or their fragments, the platelet fraction, and the monocyte-containing fraction are simultaneously passed through the channels or channel-like structures, such as This is the case for a whole blood fraction if it is obtained from the extracorporeal amount of blood as described below. It should further be understood that the procedure can be performed although not as effectively by adhering only plasma components to the walls of the flow chamber and allowing monocytes to interact with the plasma components. However, in the following, this aspect will be analyzed for a preferred embodiment, that is, where steps (i), (ii) and (iii) are carried out.

With regard to the first step, plasma components can be provided, including proteins such as fibrinogen or fibronectin, or fragments thereof such as the gamma component of fibrinogen, either as fractions obtained from the extracorporeal amount of blood sample or in purified form of other resources, for example, in the form of recombinantly expressed proteins. Although it appears that the activation of platelets by plasma proteins such as fibrinogen and fibronectin is sufficient for the recombinantly expressed forms of these proteins to be sufficient, it may be preferred to use plasma fractions that are obtained from the extracorporeal amount of blood sample and that they comprise these proteins as these plasma fractions have a more complex composition and can comprise all plasma components, which provide optimal activation of platelets.

Plasma protein fractions, plasma proteins or their fragments can be passed through the flow chamber, which can be made of plastic or non-plastic materials such as glass to adhere to channel walls or channel-like structures. It is not necessary for the plasma fractions or plasma proteins to pass through the flow chamber with a specific shear stress, such as for example a specific pressure. However, to expedite the procedure, it is envisaged to pass the plasma or plasma protein fractions through the flow chamber with a shear stress that is comparable, if not identical, to the shear stress required for activation of monocytes that it is described in more detail below. In general, the plasma fractions or plasma proteins are first pumped through the flow chamber to coat their surfaces with plasma proteins, which include fibronectin and fibrinogen. The flow rate of the plasma protein fractions, plasma proteins or fragments thereof through the flow chamber is controlled to obtain a desired level of protein adherence to plastic surfaces. If desired, the flow can be stopped for a period of time and the plasma component can "soak" the surfaces of the flow chamber. By controlling the speed and time of the pump that drives the plasma components through the flow chamber, the degree of coating can be controlled. In one approach, plasma fractions or plasma proteins are exposed to the surfaces of the flow chamber structures for a period of between about 1 and 60 min, between about 1 and about 30 min, between about 1 and about 20 min. , or between about 1 and about 10 min. To enhance adherence of plasma proteins to flow chamber surfaces, flow can be temporarily interrupted (up to approximately 60 minutes), before resuming, or flow can be reduced relative to fill rate (up to 100 ml / minute ) up to as little as 5 ml / minute, during this phase of the procedure.

A scenario can also be envisaged where a device with a flow chamber is used whose surfaces of the flow chamber have been previously coated with, for example, purified plasma proteins or fragments thereof, such as the gamma component of fibrinogen. Such pre-coated devices can be used if the entire procedure is carried out in a portable device comprising a cartridge providing the flow chamber, which is configured for, for example, single use. A scenario can also be envisaged where a device with a flow chamber is used whose surfaces of the flow chamber have been previously coated with, for example, platelet rich plasma.

After the plasma fractions or plasma proteins or their fragments have been passed through the channels or channel-like structures and the surfaces thereof have been coated with plasma proteins, the platelet fraction is passed, for example, pumping into and through channels or channel-like structures. The flow rate and residence time of platelets within the channels or channel-like structures is selected to allow the platelets to bind to plasma components or proteins or fragments thereof that have previously adhered to the channel surfaces. or canal-like structures. so that in this way they are activated.

The data presented herein suggest that platelet activation by plasma components is a sequential procedure in which inactivated platelets first bind to the gamma component of fibronectin, thereby become activated, and then can bind to the RGD (arginine , wisteria, aspartic acid) found in many plasma proteins such as fibronectin or fibrinogen. If recombinantly expressed and / or purified plasma proteins or fragments thereof are used for platelet activation, it is therefore possible to envisage coating channels or channel-like structures with at least the gamma component of fibrinogen and optionally with peptides RGD additionally. These peptide and plasma protein fragments can allow efficient platelet activation and at the same time optimal control of the coating process of channel surfaces or channel-like structures. Of course, all of these components are present if a plasma fraction obtained from the extracorporeal amount of blood is used for coating and activation.

For effective binding of platelets to plasma components and their activation in this way, the flow rate can be adjusted up or down compared to the stage of coating the plasma components, or the flow can be stopped for a period of time, to obtain the desired level of platelets bound to plasma components. Flow rates for plasma activation will typically range from about 5 ml / min to about 200 ml / min, from about 10 ml / min to about 150 ml / min, from about 10 ml / min to about 100 ml / min. , or from about 5 ml / min to about 50 ml / min. Typically, it will be desirable to allow between about 1 and 60 minutes, between about 1 and about 30 minutes, between about 1 and about 20 minutes, or between about 1 and about 10 minutes for the platelets to bind to the plasma components.

Although the shear stress does not appear to be as important for platelet activation as for monocyte activation, it may be preferable to pass the platelet fraction through the flow chamber under a shear stress of about 0.1 to about 20.0 dynes / cm2, from about 0.2 to about 15.0 dynes / cm2, from about 0.3 to about 10.0 dynes / cm2 such as from about 0.2 to about 0.4, to about 0 , 5, to about 0.6, to about 0.7, to about 0.8, to about 0.9, to about 1, to about 2, to about 3, to about 4, to about 5, or about 6 dynes / cm2. Typical flow rates of the platelet-containing fraction may range from about 5 ml / min to about 200 ml / min, from about 10 ml / min to about 150 ml / min, from about 10 ml / min to about 100 ml. / min, or from about 5 ml / min to about 50 ml / min. Flow rates will depend to some extent on the size and geometry of the flow chamber and can be used in particular if a flow chamber of the dimensions mentioned below is used. In general, flow rates will be selected to achieve the shear stress values mentioned above.

Therefore, it is contemplated to pass the platelet-containing fraction through the channels or channel-like structures at a flow rate of about 10 ml / minute to about 200 ml / minute to produce a shear stress of about 0.1 to about 10 , 0 dynes / cm2.

After the platelets have passed through the channels or channel-like structures and have been activated by the plasma proteins or fragments thereof, which have been arranged on the surfaces of the channels or channel-like structures thereof, the monocyte-containing fraction, for example the extracorporeal amount of said mammalian subject blood sample or the fraction of leukocytes or buffy coat mentioned below, which has been obtained from the extracorporeal amount of blood sample, is passed, for example, pumping into and through channels or channel-like structures, applying a shear stress. It should be understood that activation of platelets through interaction with plasma components will lead to adherence of platelets to plasma components.

It should also be understood that the same events will occur as described above if an extracorporeal amount of a mammalian blood sample comprising platelets and plasma components passes through the flow chamber. In this case, the plasma components will adhere to the walls of the flow chamber and then activate the platelets. However, in this scenario, the procedure may be less controllable and this can be accounted for by increasing the residence time of the extracorporeal amount of a mammalian blood sample comprising platelets and plasma components in the flow chamber.

It should further be noted that, instead of activated platelets, platelet-derived factors, which are sufficient to activate monocytes, can be used. These factors include, for example, fibronectin and may also include factors such as P-selectin, α5pl integrin, the C-type lectin receptor, CD61, CD36, CD47, and complement inhibitors such as CD55 and CD59, or TREM-like transcript. -1. Said platelet-derived factors can also be disposed directly on the surfaces of the flow chamber, either, for example, as mixtures of purified components or as mixtures of components obtained, for example, by lysis of platelets contained within the extracorporeal amount of a blood sample from a mammalian subject. In this case, the need, for example, to coat the surfaces of the flow chamber with plasma components can be avoided.

The data presented herein suggest that once platelets have been activated, activated platelets express proteins such as P-selectin and RGD-containing ligands, which can then interact with monocytes and activate their differentiation into immunostimulatory dendritic cells. Furthermore, it was found that the activation of monocytes and the induction of dendritic cells by activated platelets do not occur under static conditions. Rather, monocytes need to pass through the channels or channel-like structures under the application of a shear stress. Since platelets after activation require approximately 60 to approximately 120 minutes to express factors such as P-selectin, which then activates monocytes, passage of monocytes may be delayed until platelets have begun to express these factors, for example. example, from about 60 to about 120 minutes. If an extracorporeal amount of a mammalian subject blood sample comprising monocytes, platelets, and plasma components is passed through the flow chamber, this time period may have to be adjusted to longer times.

It should be understood that the interaction of monocytes with activated platelets, platelet derived factors or plasma components is not sufficient for the activation and differentiation of monocytes without the application of a shear stress at the same time.

The application of a physical force to move the monocyte-containing fraction through the flow chamber preferably means that a monocyte-containing fraction, such as the extracorporeal amount from a blood sample from a mammalian subject, moves through the chamber. flow under shear stress. Typically, the monocyte-containing fraction can be passed through the flow chamber under a shear stress of from about 0.1 to about 20.0 dynes / cm2, from about 0.2 to about 10.0 dynes / cm2, such as from about 0.2 to about 0.3, to about 0.4, to about 0.5, to about 0.6, to about 0.7, to about 0.8, to about 0.9, to about 1, to about 1.5, or to about 2 dynes / cm2. Typical flow rates of the monocyte-containing fraction may range from about 5 ml / min to about 200 ml / min, from about 10 ml / min to about 150 ml / min, from about 10 ml / min to about 100 ml. / min, or from about 5 ml / min to about 50 ml / min. Flow rates will depend to some extent on the size and geometry of the flow chamber and can be used in particular if channels or channel-like structures of the dimensions mentioned below are used. In general, flow rates will be selected to achieve the shear stress values mentioned above.

Therefore, it is contemplated to pass the monocyte-containing fraction through the channels or channel-like structures at a flow rate of from about 10 ml / minute to about 200 ml / minute to produce a shear stress of from about 0.1 to about 0 , 5 dynes / cm2. In any case, it must be verified that a cut-off voltage is generated that allows the binding of monocytes to activated platelets and the differentiation of said activated monocytes into immunostimulating DCs.

The data presented herein suggest that the monocyte / platelet interaction can be divided into short-acting interactions, which are arbitrarily defined as contact that occurs for less than 3 seconds by detection with an optical microscope, and long-acting interactions, which are defined as contact of more than 3 seconds by detection with an optical microscope. It appears that the initial short-acting interactions are mediated by P-selectin which is expressed on activated platelets. These initial contacts can then subsequently trigger long-acting interactions mediated by RGD-containing proteins expressed by activated platelets.

Monocyte activation and differentiation into immunostimulatory DCs can be positively influenced by allowing monocytes to make long-acting contacts with platelets, for example, by giving monocytes and platelets sufficient time to interact. As monocytes flow through the channels or channel-like structures, they alternately bind to and dissociate from platelets by the shear stress induced by flow through the channels or channel-like structures. The residence time of the monocyte / platelet interaction can be controlled by varying the flow rate, for example by controlling the speed of the pump. For example, the pump may initially run at a slow speed / low flow rate to enhance the monocyte / platelet interaction, and then the speed / flow rate can be increased to facilitate dissociation and harvesting of the treated monocytes from the treatment device. It appears that the adherence of monocytes to platelets can be achieved by improvement from about 0.1 to about 2 dynes / cm2, to about 0.1 to about 1 dynes / cm2, and preferably from about 0.1 to about 0 , 5 dynes / cm2, while monocyte dissociation and harvesting can be better achieved with increased shear stress levels.

It should be understood that activation of monocytes results in immobilization, for example, by interacting with activated platelets, platelet-derived factors, or plasma components. To harvest the induced immunostimulatory autologous dendritic cells, the shear stress can be increased to for example 20 dynes / cm2 and / or the immunostimulatory autologous dendritic cells can be treated with factors that allow dissociation of activated platelets, platelet-derived factors or plasma components by adding factors such as Plavix, Aspirin, or other anticoagulants.

Temperature is another factor that influences the activation of monocytes and their differentiation into immunostimulating autologous dendritic cells. The processes according to the invention can be carried out in a range from about 18 ° C to about 42 ° C, preferably in a range from about 22 ° C to about 41 ° C, and more preferably in a range from about 37 ° C to about 41 ° C.

A parameter that can also be varied to adjust the activation of monocytes is the density with which the flow chamber is covered with plasma components and, therefore, with platelets that bind to plasma components. In general, the more densely the surfaces of the flow chamber are coated with plasma components and platelets, the more efficient the activation of monocytes will be.

It has been mentioned above that platelets are activated by binding to plasma components. The term "activated platelets" according to the invention is used to refer to platelets that show increased expression of P-selectin, αIIb-p3 integrin and / or RGD-containing proteins such as fibronectin, fibrinogen or vitronectin as a consequence of binding platelets to plasma components such as fibronectin and / or fibrinogen. Expression can be determined by standard methods such as RT-PCR, immunoblotting or fAc S analysis. The term "non-activated platelets" according to the invention is used to refer to platelets in which binding to plasma proteins such as as fibronectin or fibrinogen by pre-incubating platelets with the gamma component of fibrinogen.

It has been mentioned above that monocytes become activated and begin to differentiate into immunostimulating autologous dendritic cells by binding to activated platelets under shear stress conditions. The term "activated monocytes" according to the invention is used to refer to monocytes which, when binding to activated platelets under shear stress conditions, express increased levels of the open conformation of p1-integrin and begin to express CD markers in maturation such as HLA-DR + / CD83 +. As a control to determine whether the interaction of monocytes with activated platelets leads to activation and differentiation of CD, the expression of HLA-DR + / CD83 + can be compared after the binding of monocytes to activated platelets under shear stress conditions, either in the absence of anti-P-selectin antibodies (activation) or in the presence of anti-P-selectin antibodies (control). Expression can be determined by standard procedures such as RT-PCR, immunoblotting, or FACS analysis.

After monocytes have been activated by a method according to the invention, they begin to differentiate into immunostimulating autologous dendritic cells. The term "immunostimulating autologous dendritic cells" according to the invention is used as mentioned above. These immunostimulatory autologous dendritic cells can be identified by the expression of markers described above. Immunostimulating autologous dendritic cells are distinguished in addition to immunosuppressive autologous dendritic cells or also called truncated because no change in GILZ expression is observed when autologous dendritic cells are obtained by a method according to the invention.

The experimental findings described herein immediately suggest various other embodiments of this first aspect that may provide different advantages.

The finding that the activation of monocytes and the subsequent induction of the differentiation of these monocytes into immunostimulating autologous DCs can be achieved in a miniaturized device, allows the procedure to be carried out with smaller amounts of an extracorporeal blood sample. As mentioned above, the classical ECF procedure requires the processing of 2.5 l to 6 μl of blood, which is typically obtained from patients by apheresis, such as leukapheresis, to obtain a final volume of approximately 200 ml to 500 ml that it comprises leukocytes, including monocytes as well as plasma components and platelets. However, the methods according to the invention may require substantially less blood samples, thereby avoiding the need for apheresis, such as leukapheresis or other procedures, which are a considerable burden on patients.

Thus, the present invention can be carried out without the need for apheresis, such as leukapheresis, and the entire procedure for obtaining such immunostimulating autologous dendritic cells can be carried out in a portable device.

Therefore, in an embodiment of the first aspect of the invention, which can be combined with the embodiments described above, it is contemplated to carry out the method according to the first aspect, wherein said extracorporeal amount of said mammalian subject blood it has not been obtained by apheresis such as leukapheresis.

Said extracorporeal amount of said mammalian subject blood can be between approximately 5 ml and approximately 500 ml, between approximately 10 ml and approximately 450 ml, between approximately 20 ml and approximately 400 ml, between approximately 30 ml and approximately 350 ml, between approximately 40 ml and about 300 ml, or between about 50 ml and about 200 ml or between about 50 ml and about 100 ml of extracorporeal blood from said mammalian subject to give a final volume of between about 1 ml and about 100 ml, between about 1 ml and about 50 ml, between about 1 ml and about 40 ml, or between about 1 ml and about 30 ml of an extracorporeal amount of a blood sample from a mammal.

The amount of extracorporeal blood drawn and applied to the device can be whole blood. Alternatively, said extracorporeal amount of said mammalian subject blood can be obtained by isolating leukocytes of between about 5 ml and about 500 ml, between about 10 ml and about 450 ml, between about 20 ml and about 400 ml, between about 30 ml and about 350 ml, between about 40 ml and about 300 ml, or between about 50 ml and about 200 ml, or between about 50 ml and about 100 ml of extracorporeal whole blood from said mammalian subject.

Said extracorporeal quantity of said mammalian subject blood can also be obtained by isolating buffy coats of between approximately 5 ml and approximately 500 ml, between approximately 10 ml and approximately 450 ml, between approximately 20 ml and approximately 400 ml, between approximately 30 ml and approximately 350 ml, between about 40 ml and about 300 ml, or between about 50 ml and about 200 ml or between about 50 ml and about 100 ml of extracorporeal whole blood from said mammalian subject.

In all of the cases mentioned above (whole blood, leukocyte fraction, buffy coats), said extracorporeal amount of blood will typically comprise between about 1x104 and about 1x108, such as about 5x106 mononuclear cells / ml.

One of skill in the art is familiar with how to obtain whole blood, a leukocyte fraction thereof, or a buffy coat fraction thereof (see, for example, Bruil et al., Transfusion Medicine Reviews (1995), IX (2 ), 145-166) which includes filtration, differential centrifugation. A preferred procedure is based on filters, such as are available, for example, from Pall. Such filters can be incorporated into the device so that processing of the extracorporeal sample can be performed on the portable device. As a source, for example, umbilical cord blood can also be used.

While centrifugation is used, whole blood can be obtained through a syringe with, for example, a 17 or 18 gauge needle. Such a whole blood sample can be centrifuged to remove debris and other components. The whole blood sample can then be filtered through common filters, as available from Pall.

To obtain a mononuclear leukocyte fraction, a whole blood sample can be obtained as described and the sample then applied, for example, to Ficoll-Hypaque. Subsequently, a centrifugation step is performed at, for example, from about 100g to about 200g, such as 180g, and then the mononuclear leukocyte fraction can be collected from the interface and washed with common buffers such as HBSS. The washed mononuclear leukocyte fraction can then be resuspended in serum-free cell culture medium such as RPMI-1640 medium (GIBCO). Other procedures for obtaining mononuclear leukocyte fractions include elutriation, filtration, density centrifugation, etc.

As noted above, the critical steps for induction of DC formation appear to involve the activation of platelets by plasma components and the activation of monocytes by these activated platelets. In principle, a whole blood sample could be passed through the device under shear stress. The plasma components of said sample will then bind to the surfaces of the flow chamber and allow the adherence and activation of platelets within said sample by the plasma components. The monocytes in that sample will then bind to the activated platelets and become activated themselves.

Similarly, combinations of the various components can be obtained, such as a fraction containing platelet-rich plasma, which can be obtained by centrifugation of a whole blood sample that has been obtained as described above at about 100 g to about 180 g, such as about 150 g for from about 10 minutes to about 20 minutes, such as about 15 minutes to separate debris from the whole blood sample. The platelet rich plasma layer is then collected and recentrifuged at about 700 g to about 1000 g such as about 900 g for from about 3 min to about 10 min such as about 5 min. The resulting pellet is then resuspended in serum-free cell culture medium.

However, to have the best control over the procedure, it may be desirable to pass the plasma components first through the flow chamber and allow the platelets to adhere, then the platelets, and then the fraction containing monocytes. For this approach, it may be convenient to obtain a leukocyte fraction comprising a monocyte or buffy coat fraction comprising monocytes, not comprising plasma components, and not comprising platelets. Such fractions containing plasma and platelet free monocytes can be obtained as described in the art. If the leukocyte or buffy coat fractions are obtained as described above, they will be sufficiently free of plasma or platelets for the purposes of the invention. For this approach, it may also be convenient to have platelet and / or plasma fractions.

Thus, the invention contemplates the use of platelets that have been separated from the extracorporeal amount of said mammalian subject blood before said extracorporeal amount of said mammalian subject blood is applied to said device. These platelets can then be passed through the flow chamber, which has been coated with plasma components such as fibronectin.

In another embodiment, the invention considers the use of plasma components that have been separated from the extracorporeal amount of said mammalian subject blood before said extracorporeal amount of said mammalian subject blood is applied to said device. These plasma components can then be passed through the flow chamber so that they can adhere.

Instead of using plasma components that have been obtained from the extracorporeal amount of blood, one can also use plasma components that have been isolated from other sources, such as for example, by expression of recombinant proteins. Such plasma components include fibrinogen, fibronectin, P-selectin, and fragments thereof, such as the gamma component of fibrinogen.

Although it may be preferred to use an extracorporeal amount of blood, which has not been obtained by apheresis, such as leukapheresis, the use of an extracorporeal amount of blood, which has been obtained by apheresis, such as leukapheresis, is not excluded by the invention.

Therefore, in another embodiment of the first aspect of the invention, it is contemplated to carry out the method as described above, wherein said extracorporeal amount of said mammalian subject blood has been obtained by apheresis such as leukapheresis.

Apheresis such as leukapheresis can be performed as is known in the art. Thus, an extracorporeal amount of blood, such as 2.5 l to 6 L, can be obtained from a subject and treated with conventional leukapheresis to obtain three fractions, namely plasma, platelets, and buffy coats. Plasma, which contains proteins such as fibronectin and fibrinogen, is the lightest blood fraction and therefore is the first portion of the blood to be selectively removed from the centrifuge and passed through channels or channel-like structures. . After the plasma has been pumped through the channels or channel-like structures and their surfaces have been coated with plasma proteins, the second lightest component of the leukapheresis centrifugation, the platelet fraction, is pumped through the channels or channel-like structures. The third lightest fraction to elute from the leukapheresis centrifugation is the buffy coat, which contains the white blood cells, including the blood monocytes. Next, the buffy coat that includes the monocytes is pumped through the channels or channel-like structures. A blood sample can be obtained using the Therakos device, the Spectra cell sorter (see Andreu et al., (1994), Transf. Sci., 15 (4), 443-454), or the Theraflex device from Macopharma.

Therefore, the invention in an embodiment, the invention considers the use of platelets that have been separated from the extracorporeal amount of said blood of a mammalian subject obtained by apheresis, such as leukapheresis, before said extracorporeal amount of said blood of mammalian subject is applied to said device.

In another embodiment, the invention considers the use of plasma components, which have been separated from the extracorporeal amount of said mammalian subject blood obtained by apheresis, such as leukapheresis, before said extracorporeal amount of said subject blood from mammal comprising monocytes and / or platelets is applied to said device.

Instead of using plasma components that have been obtained from the extracorporeal amount of blood, one can also use plasma components, which have been isolated from other sources, such as for example, by expression of recombinant proteins. Such plasma components include fibrinogen, fibronectin, or P-selectin. Plasma protein fragments such as the gamma component of fibrinogen corresponding to amino acids 400-411 (SEQ ID NO: 105, His-His-Leu-Gly-Gly-Ala-Lys-Gln-Ala-Gly- Asp-Val). The data presented here show that this gamma component can activate platelets. Therefore, it may be preferred to use plasma fractions, which at least, if not predominantly, comprise fibronectin. Similarly, it may be preferred to use, for example, recombinantly expressed and / or purified fibronectin or the gamma component thereof to activate platelets.

For both embodiments of the first aspect of the invention where the extracorporeal amount of blood has or has not been obtained by apheresis, such as leukapheresis, it can be considered to pass the three fractions, namely, plasma components, platelets and the fraction that contains monocytes at a time, for example, even in the form of a whole blood sample or using only pre-purified fractions of whole blood, through the flow chamber, although the sequential passage of these fractions through the flow chamber described above can provide better control over the procedure. Pre-purified whole blood fractions can be obtained, for example, by centrifuging a blood bag and extracting the supernatant, which would be enriched for white blood cells and platelets.

As mentioned, the flow rate through the flow chamber and thus the resulting shear stress will achieve the differentiation of monocytes into immunostimulating autologous dendritic cells. In addition to flow rate, the design and dimensions of the flow chamber can be varied to manipulate and even improve the application of a shear stress to the monocytes.

A device having a flow chamber with channels or channel-like structures may be suitable. Said flow chamber which has the general architecture, although in smaller dimensions, of a device used for the classical FEC procedure is represented in figure 17.

However, other geometries such as those depicted in Figure 18 a) to d) can also be used. Therefore, the findings described in the present document allow to consider flow chambers of significantly simplified geometry, which also allows to have a better control over the process in terms of turbulence and shear stress that occur during the process.

A device having a multiplicity of flow chambers may be suitable. Said flow chamber which has the general architecture, although in smaller dimensions, of a device used for the classical FEC procedure is represented in figure 17.

Typically, a flow gradient will be created in the flow chamber such as channels as the fraction containing monocytes is passed through. Monocytes will alternately bind and dissociate platelets and / or plasma components. The maturation of monocytes in immunostimulatory autologous dendritic cells is greatly enhanced by this interaction, where increased exposure to platelets and / or plasma components thus provides increased signaling of this maturation process.

To obtain as homogeneous a population of immunostimulating autologous dendritic cells as possible, it is therefore desirable that the design and dimensions of the flow chamber, such as the channels, are selected to avoid different flow zones in the flow chamber. flow.

The flow chamber, such as the channels, can in principle have any suitable cross-sectional shape for the purposes described above. Thus, they can have a rectangular, round, elliptical or other cross-sectional shape. Although the dimensions of such a flow chamber will be discussed in the following mainly with respect to a rectangular cross section, it may be preferable to use a flow chamber such as channels with an elliptical or round cross section since such cross sections would allow, for example, a more homogeneous coating with plasma components and / or more continuous flow properties with less turbulence.

If it has a rectangular cross section, the flow chamber, such as the channels, can have dimensions from about 5 pm to about 500 pm in height and from about 5 pm to about 500 pm in width. Channels or channel-like structures may also have dimensions from about 10 pm to and including about 400 pm in height and from about 10 pm to and including about 400 pm in width, from about 10 pm to and including about 300 pm in height and approximately 10 pm to and including approximately 300 pm in width, approximately 10 pm up to and including approximately 250 pm in height, and approximately 10 pm up to and including approximately 250 pm in width, approximately 10 pm up to and including approximately 100 pm in in height and from about 10 pm to and including about 100 pm in width, or from about 10 pm to and including about 50 pm in height and from about 10 pm up to and including about 50 pm in width.

If flow chambers such as channels of elliptical cross section are used, the dimensions of height and width mentioned above would have to be adapted correspondingly to allow a comparable volume. If flow chambers such as round cross-sectional channels are used, the diameter can typically be in the range of about 5 pm to and including about 500 pm, from about 10 pm to and including about 400 pm, from about 10 pm to e. including approximately 300 pm, from approximately 10 pm to and including approximately 250 pm, from about 10 pm to and including about 100 pm or from about 10 pm to and including about 50 pm.

In general, smaller dimensions are preferred for flow chambers with a particular preference for heights, widths or diameters less than 100 pm, such as 50 pm, for the reason that it is assumed that with such smaller dimensions the interaction of monocytes with platelets is more efficient and uniform and the flow properties on the surfaces and in the center of the flow chamber are more similar.

The length of the flow chamber, such as the channels and channel-shaped structures, is normally selected so that the flow chamber allows the passage of the extracorporeal blood volume. For example, the flow chamber and the device can be configured to allow passage of an overall volume of between about 1 ml and about 50 ml, between about 1 ml and about 40 ml, or between about 1 ml and about 30 ml.

The flow chamber may have internal substructures to increase the surface area or make the flow conditions less heterogeneous.

The flow chamber can be filled with particles to increase the surface area or make the flow conditions less heterogeneous.

The material of the flow chamber can be plastic or non-plastic.

If non-plastic materials are considered, glass can be used.

The surface of the chamber can be coated covalently or through adsorption.

Materials for auxiliary pipes, chambers, valves, etc. they can be selected to have reduced interactions with blood components.

The surfaces of auxiliary pipes, chambers, valves, etc. They can be treated / coated to have reduced interactions with blood components.

If plastic materials are considered, acrylics, polycarbonate, polyetherimide, polysulfone, polyphenylsulfone, styrenes, polyurethane, polyethylene, Teflon, or any other appropriate medical grade plastic can be used. In a preferred embodiment of the present invention, the flow chamber is made of an acrylic plastic.

The flow chamber can be made of a material that provides a degree of transparency such that the sample within the flow chamber, such as fractions containing monocytes, can be radiated with visible or UV light, preferably UV-A. As shown in the experiments, exposure to UV-A and 8-MOP results in increased expression of GILZ and, therefore, activation and differentiation of monocytes into immunosuppressive autologous dendritic cells. Therefore, in general, exposure to light, such as UV-A and DNA crosslinking agents, such as 8-MOP, should be avoided when producing immunostimulating autologous dendritic cells.

However, once monocytes have advanced through the maturation pathway sufficiently that immunostimulating autologous dendritic cells have formed as can be determined by the molecular markers mentioned above, one can envision administering DNA crosslinking agents such as 8 -MOP and exposing the immunostimulatory autologous dendritic cells to, for example, UV-A to cause apoptosis of other cells in the extracorporeal blood sample. Said cells can be cytotoxic T lymphocytes, virus-infected cells or bacterial cells. Apoptosis of these cells can lead to the release of circulating antigens. Immunostimulating autologous dendritic cells can then take up and process these antigens to form immunostimulatory autologous antigen presenting cells. These immunostimulating autologous antigen presenting cells can then, for example, be reintroduced into the respective individual to elicit an immune response against the respective tumor, viral or bacterial antigens. Once immunostimulatory autologous dendritic cells have been formed, tumor cells, bacterial cells or virus-infected cells of said individual can also be introduced separately into the flow chamber and cause apoptosis of these cells, for example, by additionally adding cross-linking agents. crossing DNA such as 8-MOP and radiating the mixture of immunostimulating autologous dendritic cells and tumor cells, bacterial cells, or virus-infected cells to cause apoptosis of tumor cells, bacterial cells, or virus-infected cells so that they can form immunostimulating antigen presenting cells. It is for these embodiments that a flow chamber design that allows exposure to light such as UV-A is contemplated.

A typical flow chamber can have the geometry depicted in FIG. 19A). The flow path has dimensions of 20mm by 80mm. The chamber is made of polystyrene, PET (polyethylene tephthalate), PMMA (poly (methyl methacrylate)), and silicon. A blood sample can be centrifuged at low speed through a Ficoll gradient to obtain, for example, 8 ml of sample with a white blood cell concentration of, for example, 1010 cells / ml. The chamber may be pre-coated with platelet rich plasma. The sample can be passed through the chamber at approximately 0.028 Pa for approximately ... min. The chamber can then be washed with approximately 3 ml of RPMI at 0.028 Pa. A second wash can be performed with 30 55 ml of RPMI at approximately 1.2 Pa. The harvested activated monocytes will then be pooled and used for further analysis .

Once immunostimulatory autologous dendritic cells have been obtained by methods according to the invention, they can generally be further processed for specific purposes. These newly formed immunostimulatory dendritic cells can be incubated, for example, under standard conditions to allow their maturation to complete. The cultivation of these immunostimulating dendritic cells can be carried out under standard conditions, for example, at 37 ° C and 5% CO ² in standard media for the culture of human cells such as RPMI-1640 medium (obtainable, for example, from GIBCO), supplemented with 15% AB serum (available, for example, from Gemini Bio-Products).

In this way, mature immunostimulating dendritic cells can be obtained without the need for rather expensive cytokine cocktails to induce monocyte differentiation into DCs. Although not necessary, it may be considered to grow such immunostimulatory dendritic cells in a culture medium buffered with one or more cytokines, such as GM-CSF and IL-4, during the incubation period. Ripening cocktails (which typically consist of combinations of ligands such as CD40L, cytokines such as interferon gamma, TNF alpha, interleukin 1 or prostaglandin E2 or the factors mentioned above) can also be added. In one aspect, immunostimulating dendritic cells are preferentially produced against immunosuppressive dendritic cells by culturing the dendritic cells for extended periods of time, such as, for example, at least 1, at least 2, at least 3, at least 4, or at least 5 days. This can help the initially formed immunostimulating dendritic cells to further advance their maturation pathway. However, according to the invention, immunostimulatory dendritic cells are obtained without cytokine cocktails to induce monocyte differentiation into DC.

Immunostimulatory dendritic cells in accordance with the present invention can be tested for functionality in assays as described herein. For example, the assay described in Bioley et al., The Journal of Immunology 2006), 177: 6769-6779 can be adapted. In such a tailored assay, immunostimulating dendritic cells, which are obtained by the methods disclosed herein, for example, passing white blood cells as described above through a device depicted in FIG. 19, are incubated together with CD4 + and CD8 + lymphocytes from the same donor and the peptide Melan-A / MART-1 ^26-35 , described by Bioley et al. The detection of CD4 + lymphocytes positive for Melan-A / MART-126-35 and CD8 + cells allows to confirm the functional immunostimulatory dendritic cells that are obtained according to the present invention.

In addition, said immunostimulatory dendritic cells can then be manipulated ex vivo, prior to re-administration to the subject, to suit the desired therapeutic purpose.

Thus, before being re-administered to the subject, said immunostimulating dendritic cells can be processed, for example, ex vivo, such as loading them with immunogenic antigens, for example those expressed on apoptotic tumor cells or pathogenic infectious agents, or enhancing their maturation to increase its efficiency in immunotherapy against cancer. This will result in the immunostimulating antigen presenting cells displaying the antigen on their surface. One of the most preferred embodiments of the present invention contemplates separating as much as possible the generation of immunostimulating dendritic cells as described herein, the generation of disease antigens, and the loading of these antigen-presenting cells with the antigens. of the illness. Thus, apart from, for example, in FEC, these different procedures do not occur continuously, for example, by avoiding the application of photoactivatable agents such as 8-MOP and UVA. In contrast, immunostimulatory antigen presenting cells, such as dendritic cells, are formed from monocytes by applying shear stress in the absence of, for example, 8-MOP and UVA and cytokine cocktails. These immunostimulating dendritic cells can then be incubated in conjunction with separately obtained disease antigens to effect efficient loading of the immunostimulating dendritic cells and presentation of the antigens on their surface. It is the information provided by the findings in this document that allows us to separate the multiple procedures that occur during ECF and to adjust the generation of immunostimulatory dendritic cells, for example, avoiding or reducing the formation of immunosuppressive dendritic cells.

Immunostimulatory dendritic cells can be loaded in particular with pathogens to produce antigen-presenting dendritic cells which, when reintroduced into the subject, can launch an immune response against pathogenic genes.

As used herein, the term "pathogens" refers to agents that are essential to causing a disease state in a subject. For example, immunostimulating dendritic cells can be loaded with antigens, which are known to be expressed in cancer tissue. To this end, these antigens can be expressed on immunostimulatory dendritic cells such that they are present on MHC class 1 and MHC class 2 molecules. By loading immunostimulatory dendritic cells with such antigens, subjects can be vaccinated against subsequent onset, for example, of cancer or infection. If an antigen that is already expressed in cancer tissue obtained from the subject from which the extracorporeal amount of blood was drawn is used to generate immunostimulating dendritic cells, reintroduced antigen-loaded immunostimulatory antigen presenting cells can initiate an immune response against cancer.

In certain circumstances, these pathogens are disease-causing cells that may be circulating in the bloodstream, thus making them easily accessible for extracorporeal manipulations and treatments. Examples of such disease-causing cells include, for example, malignant T lymphocytes, malignant B lymphocytes, and white or red blood cells infected by viruses or bacteria that can harbor or express microbial (eg, viral, bacterial, fungal, mycobacterial peptides or proteins. , protozoa) or other molecules associated with pathogens. Exemplary categories of diseases that give rise to disease-causing cells include lymphoproliferative disorders such as leukemia, lymphoma, and myeloma, as well as infections including malaria, human immunodeficiency virus (HIV), Epstein-Barr virus (EBV), cytomegalovirus ( CMV), hepatitis B virus (HBV), hepatitis C virus (HCV), Lyme disease, leprosy, tuberculosis, and other blood-borne pathogens.

Other disease-causing cells include those isolated from surgically removed samples of solid tumors, such as cancers of the lung, colon, brain, kidney, or skin. These cells can be manipulated extracorporeally analogously to blood leukocytes, after suspending or propagating them in tissue culture. Alternatively, in some cases, it has been shown that the circulating blood of patients with solid tumors may contain malignant cells that have shed the tumors and entered the circulation. These circulating tumor cells can provide an easily accessible source of cancer cells, which can be isolated, apoptosis, and phagocytosed by dendritic cells according to the method described and claimed herein.

Pathogens can also be obtained from said disease-causing cells, the apoptosis of which has been caused for example by cytotoxic agents. It should be understood that apoptotic cells can send different signals through effector agents depending on whether they are derived from normal or abnormal cells, such as healthy or malignant cells. The combination and culturing of immunostimulating dendritic cells with such apoptotic cells can also be used to load immunostimulating dendritic cells with disease-causing antigens and generate immunostimulating antigen presenting cells.

In addition to disease-causing cells, pathogens within the scope of the invention further include microbes, such as bacteria, fungi, and viruses, that express disease-associated antigens. It should be understood that viruses can be engineered to be "incomplete", that is, to produce distinctive disease-causing antigens without being able to function as an actual infectious agent, and that such "incomplete" viruses fall within the meaning of the term "agents. pathogens "as used herein.

Immunogenic antigens can also be obtained by treating cancer samples or cancer cells with agents known to induce immunogenic antigens such as Bortezomib.

Cancers that can be treated in particular by the approaches described above include CTCL, melanoma, prostate cancer, CCECC since, for example, disease antigens of some of these diseases are known or since, for example, models can be used initially. animals from some of these diseases.

As mentioned above, by loading immunostimulatory autologous dendritic cells obtainable by the methods described herein with antigens, autologous immunostimulatory antigen presenting cells can be produced.

To avoid, for example, protein degradation of the administered antigen and inefficient processing of soluble antigens by dendritic cells resulting in deficient T-cell responses, it is contemplated to enhance the formation of autologous immunostimulatory antigen-presenting cells by encapsulating antigens. in polymeric nanoparticles (NP), which can be made of biodegradable polymers such as polylactic acid (Waeckerle-Men et al., Adv Drug Deliv Rev (2005), 57: 475-82). Such NPs can be further modified with DEC-205 targeting moieties such as an anti-DEC-205 antibody to enhance receptor-mediated endocytosis and antigen presentation.

Thus, the present disclosure contemplates the use of encapsulation of antigens in polymeric nanoparticles to optionally further enhance the formation of autologous immunostimulatory antigen-presenting dendritic cells.

The immunostimulatory dendritic cells obtained according to the invention and the pathogens are incubated for a period of time sufficient to maximize the number of functional antigen-presenting dendritic cells in the incubated cell population. Typically, the treated blood cell concentrate and pathogens are incubated for a period of about 1 to about 24 hours, where the preferred incubation time is extended for a period of about 12 to about 24 hours. Additional incubation time may be required to fully mature the loaded immunostimulatory antigen presenting cells prior to reintroduction into the subject. Preferably, the blood cell concentrate and the pathogens are incubated at a temperature between 35 ° C and 40 ° C. In a particularly preferred embodiment, the incubation is carried out at approximately 37 ° C.

Inducing monocyte differentiation according to the procedure described above provides immunostimulatory dendritic cells in numbers that equal or exceed the numbers of dendritic cells that are obtained by costly and laborious culturing of leukocytes in the presence of cytokines such as GM-CSF and IL-4. for a couple of days. The large number of functional dendritic cells generated by the method described above provides a rapid means of presenting selected material, such as, for example, apoptotic cells, pathogens, antigens, plasmids, DNA, or a combination thereof, and thus leads to effective immunotherapy. Antigen preparations selected to elicit a particular immune response can be derived, for example, from tumors, non-malignant disease-causing cells, or microbes such as bacteria, viruses, and fungi. The antigen-loaded dendritic cells can be used as immunogens by reinfusing the DCs into the subject or by administering the cells in another manner according to known procedures to elicit an immune response, such as subcutaneous, intradermal, or intramuscular injection. As described below, it is also possible to generate antigen-loaded dendritic cells by treating and co-incubating monocytes and pathogens, which can express disease-associated antigens.

As mentioned above, immunostimulatory dendritic cells can be obtained by a process according to the invention in the absence of a photoactivatable agent and without exposure to light such as visible light and preferably UV-A.

Therefore, the present invention aims to obtain functionally and maturately synchronized autologous immunostimulatory dendritic cells individual specific.

In a second aspect, the present disclosure relates to autologous immunostimulatory dendritic cells obtainable by a method described herein, preferably for use in immunization against tumor antigens, viral antigens, bacterial antigens, or fungal antigens.

The invention is now described with respect to some specific examples which, however, are for illustrative purposes and are not to be construed in a limiting manner.

Experiments

Experiment 1 - Platelet shear stress and activation to induce monocyte activation Materials and procedures

Obtaining leukocytes and platelets

All samples were obtained from young, healthy subjects who were not taking medications, including acetylsalicylic acid, which is known to influence platelet function. Samples were obtained following the guidelines of the Yale Human Research Review Commission and informed consent was given in accordance with the Declaration of Helsinki. Peripheral blood samples were drawn through a 19 gauge needle from the antecubital vein with syringes containing heparin, then applied to Ficoll-Hypaque (Gallard-Schlessinger, Carle Place, NY). After centrifugation at 180 g, the interface containing the fraction of mononuclear leukocytes was collected and washed twice in HBSS, then resuspended in RPMI-1640 medium (GIBCO) at a final concentration of 5 x 106 mononuclear cells / ml. Cells were used within one hour of being obtained.

Platelet Rich Plasma Preparation

Whole blood was centrifuged at 150g for 15 minutes at room temperature. The platelet rich plasma (PRP) layer was collected and centrifuged at 900g for 5 minutes, and the platelet pellet was resuspended in RPMI 1640 at the desired concentration.

Preparation of parallel plates

Two parallel plate flow chambers were used to model the flow dynamics of the FEC. Experiments involving post-flux cell phenotype assessment were performed using the larger Glycotech system (Glycotech, Rockville, MD). This system consisted of a volumetric flow path measuring 20,000 x 10,000 x 254 microns (length x width x height). The lower plate of this system consisted of a 15 mm Petri dish (BD Biosciences, Durham, NC) separated by a gasket and connected under vacuum to an acrylic flow platform, which formed the upper plate. For experiments that required plates to be pre-coated with platelets, before assembling the flow chamber, 20 drops of the desired concentration of PRP were placed in the center of the Petri dish and the platelets were allowed to stand for 20 minutes at room temperature. The Petri dish was washed twice with 2 ml of RPMI and then the flow chamber was assembled.

For experiments that did not require harvesting and post-flow phenotyping of cells, Vena8 biochips (Cellix Ltd, Dublin, Ireland) were used to generate laminar flow. The volumetric flow path for one channel of the Vena8 biochips measured 20,000 x 400 x 100 microns (length x width x height). The protein coating of these chips is described in the appropriate section below.

Experiments using parallel plates

The parallel plate flow chamber was mounted on the stage of a phase contrast optical microscope (CK40, Olympus, Japan) with a 10x objective. All batches were run at room temperature. A uniform laminar flow field was simulated using a syringe pump (KD Scientific, New Hope, PA) that can generate nearly constant volumetric flow rates. The configuration components were designed to minimize tubing. Prior to infusing the cell suspensions through the plates, the system was washed with 5 ml of RPMI at a flow rate that produced a wall shear stress of approximately 1 dyne / cm2. The cell suspensions of interest were then passed through the chamber at a fixed flow rate and wall shear stress. All experiments were viewed in real time, recorded at 15.2 frames per second using a DP 200 digital camera and computer program (DeltaPix, Maalov, Denmark), and analyzed using Image J computer program (NIH).

I grow overnight

When overnight culture was necessary, cells were centrifuged and resuspended in RPMI-1640 medium (GIBCO), supplemented with 15% AB serum (Gemini Bio-Products) at a final concentration of 5 x 106 cells / ml. Cells were grown overnight for 18 hours in 12-well polystyrene tissue culture plates (2 ml per well) at 37 ° C in 5% CO ² .

Immunophenotyping

Monoclonal antibodies for immunophenotyping included CD14 (LPS receptor; monocytes), CD11c (integrin subunit; monocytes and CDs), HLA-D ^r (MHC class II molecule), CD83 (CD marker), CD62p (P- selectin; activated platelets), and CD61 (integrin subunit; platelets). Antibodies were obtained from Beckman Coulter (CD14, CD11c, HlADR, CD83) or Sigma (CD62p, Cd61) and used at their predetermined optimal dilutions. Background staining with appropriate isotype controls was established, and immunofluorescence was analyzed using an FC500 flow cytometer (Beckman Coulter). Two-color membrane staining was performed by adding the predetermined optimal concentrations of both antibodies directly conjugated to FITC or PE and incubating for 20 minutes at 4 ° C, followed by washing to remove unbound antibodies. Combined membrane and cytoplasm staining was performed following the manufacturer's instructions for cell fixation and permeabilization (Intraprep kit, Beckman Coulter).

Ultra-fast quantitative PCR

Gene expression was compared between cells exposed during flow through the parallel plates at low levels (10 5 / low power field [lpf]) versus high levels (102 ± 32 / lpf) of platelets, followed by culture overnight. Cellular RNA was isolated using RNeasy Mini Kit columns with DNase I treatment on the column (QIAGEN). The yield and purity of RNA were measured using a NanoDrop ND-1000 spectrophotometer and an Agilent 2100 bioanalyzer. Reverse transcription of RNA to cDNA was performed using the high throughput cDNA reverse transcription kit (Applied Biosystems). Reverse transcription was performed in a 96-well thermal cycler (MJ Research ^Pt C-200) under the following conditions: 25 ° C, 10 minutes, 37 ° C, 120 minutes, 85 ° C, 5 seconds. TaqMan flash PCR was used to detect DC-LAMP, CD40, ADAM-DEC, Lox1, CCR7, CD80, CD83, CD86, FPRL2, and GPNMb transcripts. The primers and probes for each sequence were obtained as Taqman gene expression catalog assays (Applied Biosystems). HPRT1 was used as the reference gene. Platelet co-cultures with monocytes

Monocyte co-culture experiments with additional platelets were performed as described in the Overnight Culture section, with some necessary modifications. After Ficoll-Hypaque separation, mononuclear cells were resuspended in 30% AB serum in RMPI at a final concentration of 10 x 10 6 cells / ml, of which 1 ml was distributed to each well of a 16-well plate. An additional 1 ml of platelets (suspended in RPMI, at 2 times the desired final concentration) or RPMI without platelets was then added to each well. To activate platelets, 500 µl containing 2 units of thrombin was added to half of the wells, and 500 µl of RPMI was added to the others to balance the volume. The cells were then incubated as described above.

Platelet adhesion studies

Platelet adhesion experiments were performed using the Vena8 flow chamber described above. Fibrinogen and fibronectin (Sigma) were dissolved in PBS to a final concentration of 200 mcg / ml. The channels of the Vena8 chips were incubated at room temperature in a humidified chamber for 2 hours with the protein solution, autologous plasma or PBS alone. The channels were washed with 5 times the volume of RPMI. Platelet rich plasma was then perfused through the protein coated channel at the indicated shear voltage which was kept constant. For each channel, static images were obtained exactly 90 seconds after starting the experiment in 4 predefined low-power fields, located along the flow path (the fields were centered at 2500, 7500, 12500 and 17500 microns from the point infusion start).

Some experiments included pretreatment of platelet-rich plasma with protein fragments prior to infusion through the channels. Small RGD peptides, containing the amino acid sequence Arg-Gly-Asp-Ser; DRG peptides containing the amino acid sequence Ser-Asp-Gly-Arg; or fibrinogen fragment 400-411, which contained the amino acid sequence His-His-Leu-Gly-Gly-Ala-Lys-Gln-Ala-Gly-Asp-Val, were incubated at a concentration of 2 mM with PRP for 20 minutes at room temperature. The PRP was then perfused through the channels as previously described.

Receptor and ligand studies

Platelet-coated Vena8 channels were pretreated with 40 pg / ml of anti-P-selectin antibodies (R&D Systems) or 40 pg / ml of an isotype control for 30 minutes at room temperature, then washed with 5 times the volume. by RPMI. Mononuclear cell suspensions were pretreated with RGD or DGR peptides at a concentration of 2.5 mM. Video samples with a duration of 400 frames (26.3 seconds) were recorded 60 seconds after the start of the flow using a low power field spanning 400 microns and centered at 7500 microns from the start point of the flow.

The conformation of the integrin p-1 was evaluated using the Glycotech flow chamber. Platelet-coated 15 mm Petri dishes (described above) were pretreated with 40 pg / ml anti-P-selectin antibody or an isotype control for 20 minutes at room temperature, then washed with 5 times the volume of RPMI . Immediately after platelets were perfused, cells were immunophenotyped with anti-CD29 HUTS-21 antibody (BD Biosciences), an antibody that specifically binds to the active (open) conformation of pl integrins.

Results

Flowing monocytes transiently interact with immobilized platelets

Initially, ECF was developed as a means to allow extracorporeal exposure of pathogenic leukocytes to the chemotherapeutic 8-methoxypsoralen (8-MOP), a DNA cross-linking drug activated by ultraviolet A (UVA) light. Thus, ECF involves the flow of leukapheresis-treated blood between large parallel plates of clear plastic 1 mm apart. To allow detailed analysis of the flow dynamics involved during FEC, regardless of exposure to UVA / 8-MOP, the FEC flow conditions were reproduced using miniature parallel plates with a surface area of only 0.8 mm2, separated by 100 microns. This model allowed visualization using digital microscopy. Studies using the model revealed the following sequence (determined by video analysis): initial adherence of platelets from the flow stream to the plate, followed by transient binding of passed monocytes to the immobilized platelets.

CD induction correlates with the number of monocyte / platelet interactions

Based on the initial qualitative observations described above, it was hypothesized that platelets induced monocyte differentiation into CD under flow conditions. To test the influence of platelets on the differentiation of monocytes into DC, monocytes were passed between parallel plates previously coated with autologous platelets at a low density (10 ± 5 / low-power field [lpf]), medium (44 ± 20 / lpf) and high (102 ± 32 / lpf). The cells were passed through the plates at a flow rate that produced a wall shear stress of 0.5 dynes / cm2, analogous to the wall shear stress of postcapillary venules. The number of monocyte / platelet interactions per unit time increased in proportion to the increase in the density of platelets (determined by video analysis). An average of 52.3 15 monocyte / platelet interactions per lpf per second was observed with the high density plate, falling to 18.3 ± 14 and 3.4 ± 1 interactions per second with the medium and low density plates, respectively. (figure 1a).

After overnight incubation, a correlation was found between the percentage of cells that developed a CD phenotype and the frequency of physical monocyte / platelet interactions observed the day before (Figure 1b). An increasing number of monocyte / platelet interactions correlated with an increased proportion of cells expressing markers consistent with membrane differentiation into CD, HLA-DR, and CD83. An average of 14.2% of monocytes exposed to the high platelet density coated plate were HLA-DR + / CD83 + after overnight incubation, compared to 4.9% and 0.8% of those monocytes exposed to plates coated with medium and low platelet levels, respectively. Monocyte exposure to platelets results in changes in gene expression

To complement the described changes in the phenotype of monocytes observed after exposure to platelets, RT-PCR was performed to evaluate changes in gene expression. Monocytes were passed through parallel plates coated with high or low platelet densities as described in the previous section. After overnight incubation, RNA was extracted and RT-PCR was performed to determine the level of expression of 10 genes associated with CD (Figure 2). CD40, a costimulatory molecule with known expression in mature DCs (Cella et al., 1996, see reference list), was found to be upregulated by more than 567% in monocytes exposed to high platelet densities relative to monocytes. exposed to low levels. LAMP3, a specific marker of CD differentiation (de Saint-Vis et al., 1998, see reference list), was 398% up-regulated. CD80 is a costimulatory molecule known to be upregulated upon activation of APCs (Slavik et al., 1999, see reference list), it was upregulated by 220% in monocytes exposed to high levels of platelets. CCR7, a chemokine receptor known to play a role in CD migration to lymphatic organs, was upregulated by 376%. LOX1, c D83, CCR7, and ADAM-DEC, all genes associated with CD (Berger et al., 2010, see reference list), were also up-regulated in monocytes exposed to high levels of platelets. FPRL2, GPNMB, and CD86 were down-regulated in monocytes exposed to high levels of platelets. FPRL2 is a receptor that when activated is known to inhibit CD maturation (Kang et al., 2005, see reference list) GPNMB is a protein that participates in the decrease in cytokine production (Ripoll et al., 2007 , see list of references); CD86 is a costimulatory molecule expressed by CPA. CD induction in the presence of platelets does not occur under static conditions

Platelets could potentially influence monocytes through direct receptor / ligand interaction, or through cytokines and other secreted mediators. To determine whether platelets induction of monocyte differentiation into DCs requires flow dynamics, we tested the role of platelets under static conditions. Monocytes with low (<50,000 / mm3), medium (100-200,000 / mm3) and high (> 400,000 / mm3) platelet concentrations were co-cultured with platelets in an inactive or active state (induced by the addition of thrombin). After incubation overnight under static conditions (cutoff voltage = 0), we found that neither activated nor non-activated platelets were able to induce differentiation of monocytes into CD in the absence of flow (see Figure 3).

Platelets suspended in flow bind to serum proteins adsorbed on the plate

Several proteins abundantly present in plasma, including fibronectin and fibrinogen, are well known to adsorb onto glass and plastic surfaces; therefore, the contribution of adherent plasma proteins to platelet adhesion and activation was evaluated. Parallel plates were precoated with fibrinogen, fibronectin, plasma, or saline. Unactivated platelets were then passed at speeds producing wall shear stresses in the range of 0.2 to 6.0 dynes / cm2. Platelets adhered at higher concentrations to fibrinogen-coated plates (Figure 4). Adhesion to fibronectin-coated, plasma-coated, and uncoated plates was also observed, but to a significantly lesser extent (p <0.05). In the absence of flow, platelet adherence was equivalent on all protein substrates. Both fibrinogen and fibronectin contain segments with the amino acid sequence arginine (R) -glycine (G) -aspartate (D), RGD. RGD segments are known to interact with many integrin receptors, in particular the I / A domain of beta subunits, which are exposed when the integrins are in the active conformation (Xiong et al., 2002, see references). In experiments using fibrinogen-coated plates, platelet adhesion was not significantly altered by preincubation of platelets with RGD peptides; however, adhesion was significantly decreased (p <0.05) by preincubation of platelets with peptide fragments corresponding to amino acids 400-411 of fibrinogen, the gamma component of the protein (Figure 5a). In experiments using fibronectin-coated plates, pre-incubating platelets with RGD peptides significantly decreased adhesion, while pre-incubating platelets with peptide fragments corresponding to amino acids 400-411 of fibrinogen had no effect (Figure 5b). Interestingly, it should be noted that, unlike the I / A domain of integrins, which is known to interact with RGD domains of proteins, the region of integrin that has been found to interact with the gamma component of fibrinogen is exposed in the inactive state of integrin (Weisel et al., 1992, see references). Therefore, these data suggest that flux-inactivated platelets bind to the gamma component of fibrinogen-coated plates. The potential for inactive platelets to bind to fibrinogen may account for the higher level of platelet adhesion observed in fibrinogen-coated plates discussed in the previous paragraph.

Platelets are activated by adhesion to the plate

Platelets circulate physiologically in an inactive state, with a series of proteins stored in intracellular granules. Upon encountering stimuli such as damaged endothelium or thrombin, platelets are activated and almost instantaneously translocate these intracellular proteins to the plasma membrane (Kaplan et al., 1979, see references). It was postulated that the adhesion of platelets to the absorbed protein / plastic plate caused activation of platelets similar to that caused by well-known stimuli. To test this hypothesis, the surface expression of P-selectin, a well-known marker of platelet activation, was evaluated before and after adhesion. Before adhesion, it was found that 6 ± 3% of platelets expressed P-selectin, with a mean fluorescence intensity (MFI) of 12.4 ± 6.9; after adherence, the positivity for P-selectin increased to 64 ± 13% (MFI: 98.2 ± 14). In the positive control, thrombin-activated platelets, 71 ± 18% were P-selectin positive (MFI; 108.3 23). The expression of P-selectin was further evaluated in the 30, 60 and 90 minutes following the adhesion of the platelets; P-selectin expression remained stable at all time points, with 72 ± 11% P-selectin positive platelets 90 minutes after adhesion, indicating that platelets remain in an active state for the duration of the procedure . Similar trends were found in the evaluation of aIIb-p3, a fibrinogen-binding integrin, with an increase in the expression of this protein on the surface from 4 ± 3% before adhesion, to 49-18% after adhesion.

Monocytes interact with P-selectin and RGD-containing ligands expressed on activated platelets

The monocyte / platelet interactions observed on video were divided into two categories: (1) short-acting, which was arbitrarily defined as contact that occurs for less than 3 seconds (46 frames), and (2) long-acting, which is defined as contact for more than 3 seconds, including stable bonding. As it had previously been determined that platelets in the FEC system were in the activated state, and that activated platelets express a number of proteins including P-selectin and ^r G ^d containing proteins (e.g., fibronectin, fibrinogen, and vitronectin), An attempt was made to determine the participation, if any, of these proteins in interactions of short or long duration. Plates were precoated with platelets and four conditions were tested: (1) platelets pretreated with an irrelevant isotype control and untreated monocytes (P + RGD +); (2) platelets pretreated with an irrelevant isotype control, and monocytes preincubated with RGD peptides (P + RGD-); (3) platelets pretreated with anti-P-selectin antibody and untreated monocytes (P-RGD +); (4) platelets pretreated with anti-P-selectin antibody and monocytes pretreated with RGD peptides (P-RGD-). It was assumed that pretreatment of monocytes with RGD peptides should result in a decreased number of free RGD recognition receptors available to interact with platelet-expressed RGD-containing proteins. Thus, the four conditions tested represent each potential interaction permutation with two platelet ligands, P-selectin and RGD-containing proteins. As shown in Figure 6, both short-acting and long-acting interactions were maximal when neither RGD nor P-selectin were blocked (P + RGD +); the level of interaction in all other conditions was expressed as a percentage of this maximum. Blocking with anti-P-selectin alone (P-RGD +) resulted in a decrease in short and prolonged monocyte / platelet interactions to almost zero (p <0.01; Figure 6, also confirmed by video analysis). In contrast, blocking RGD alone (P + RGD-) did not significantly alter the number of interactions of short duration, but decreased monocyte / platelet interactions of long duration by 44% (p <0.05; Figure 6) . The simultaneous blocking of P-selectin and RGD (P-RGD-) resulted in a pattern similar to that observed when only P-selectin was blocked, where the long and short duration interactions were reduced to almost zero. The most appropriate conclusions, based on the pattern of interactions observed in each of the four conditions, are the following: (1) P-selectin is predominantly responsible for the interactions of short duration; (2) RGD-containing proteins expressed by platelets are involved in long-duration interactions, but not short-lived interactions; (3) The interaction of monocytes with P-selectin must occur upstream of the interaction of monocytes with platelet-expressed RGD-containing proteins. This latter conclusion is based on the observation that P-RGD + conditions decreased short- and long-duration interactions to almost zero, while P + RGD conditions only decreased long-duration interactions. If the interactions were not sequential, the P-RGD + conditions should have produced similar results to P + RGD + in terms of long-duration interactions. Furthermore, the order of the interactions, that is, that P-selectin acts upstream of the RGD interactions, is evident from the finding that the P + RGD- conditions only influenced the long-duration interactions, whereas P-RGD + conditions produced similar results to P-RGD-.

Exposure of monocytes to P-selectin results in the downstream activation of monocyte integrins Integrin receptors, in their open conformation, are known to interact with RGD-containing ligands (Ruoslathi et al., 1996, see references). Using an antibody that recognizes an epitope exposed only when the pl integrin is in its open conformation, we assessed the conformation of the monocyte integrins before and after flow through the model. Figure 7 shows that, as the number of short acting monocyte / platelet interactions increased, there was a corresponding increase in the percentage of monocytes expressing integrins in their open conformation immediately after flow.

The black line shows that an average of 71% of the monocytes that had received a high number of platelet interactions (> 61 ± 19 / lpf xs) expressed p1 in the active form, compared with 9% of the monocytes that had received a low number of platelet interactions (<5.1 2 / lpf xs). These results were not significantly affected by pretreatment of adhered platelets with an irrelevant isotype control (gray line). In contrast, pretreatment of platelets with anti-P-selectin reduced monocyte / platelet interactions to almost zero, and monocytes that emerged from the flow under these conditions (dashed line) showed low levels of active p1 integrins, regardless of density. platelets to which they were exposed. Notably, all cell populations prior to plating demonstrated similar low levels of integrin activation at baseline (<10%); therefore, the observed differences in short-lived monocyte / platelet interactions were not the result of differences in integrin conformation prior to flux.

Exposure of monocytes to P-selectin is required for differentiation into CD

Since monocyte / platelet interactions are dependent on platelet P-selectin, we set out to determine whether there was a relationship between the exposure of monocytes to P-selectin at time 0, and the phenotype subsequently developed by the monocyte after overnight incubation. , 18 hour time (figure 8). Monocytes were passed through parallel plates coated with high densities (108 ± 36 / lpf) of untreated (unblocked) or pretreated platelets with anti-P-selectin antibody or an isotype control. 15.5 ± 4% of monocytes exposed to unblocked platelets were transformed into membrane HLA-DR + / CD83 + (maturing CD markers) after overnight incubation, and 13 ± 4% of exposed to platelets blocked with the irrelevant isotype control. In contrast, only 3 ± 2% of monocytes exposed to platelets blocked with anti-P-selectin were transformed into HLA-DR + / CD83 + after overnight incubation.

Experiment 2 - identification of molecular markers of immunosuppressive dendritic cells Materials and procedures

Collection of samples and enrichment of monocytes

Peripheral blood samples were obtained from healthy subjects following the guidelines of the Yale Human Research Review Commission and informed consent was given in accordance with the Declaration of Helsinki. PBMC were isolated by centrifugation on a Ficoll-Hypaque gradient (Isolymph, CTL Scientific). Freshly isolated PBMC were enriched for monocytes by: 1) adherence to plastic for dexamethasone dose titration experiments (purity: 71.6 ± 5.6% CD14 +); 2) positive selection with magnetic microspheres for CD14 (Miltenyi Biotec) for PUVA dose titration experiments (purity: 88.1 ± 3.5% CD14 +), and; 3) Monocyte isolation kit II (Miltenyi Biotec) for LPS stimulation experiments (purity: 83.8 ± 3.8% CD14 +).

Generation of monocyte-derived CDs ( CDMo)

Monocytes were grown at a density of 5 x 106 cells / ml in 6 and 12-well polystyrene tissue culture plates at 37 ° C and 5% CO ² in RPMI-1640 (Gibco) supplemented with 15% AB serum heat inactivated (Gemini) and 1% penicillin / streptomycin (now called complete medium). 800 IU / ml of recombinant human GM-CSF (R&D Systems) and 1000 IU / ml of recombinant human IL-4 (R&D Systems) were added to cultures for 36 h to induce monocyte differentiation into DC as described.

Treatment with 8-MOP and UVA light

The cultures were incubated with 8-MOP (Uvadex, 20 pg / ml) for 30 minutes in the dark, and then irradiated with a desktop UVA light box containing a series of 12 linear fluorescent tubes. The tubes emitted UVA light in the 320-400 nm range. UVA irradiance (power, W / m2) was measured using a photodiode. Given a measured irradiance and the absorption properties of the various components of the system, it was possible to determine the time (s) of cell exposure necessary to deliver a given dose of UVA radiation (J / cm2).

CDMo / lymphocyte co-cultures

Non-adherent cells (purity: 66.0 ± 4.5% CD3 +) removed during adhesion to plastic will now be generally referred to as lymphocytes. Lymphocytes were treated with 8-MOP (100 ng / ml) and UVA (1 J / cm2), washed with PBS and co-cultured in complete media at 37 ° C and 5% CO ² with CDMo treated with PUVA or not treated in a ratio of 5 or 10 lymphocytes for each CDMo. As a positive control group, CDMo treated for 24 h with 100 nM dexamethasone (Sigma) were used. After 24 h, the cells were harvested and the MoDCs were purified again. To ensure that RNA was not isolated from lymphocytes in significant quantities, it was critical to re-purify the CDMo from all cultures using positive selection with magnetic microspheres for CD11c (Miltenyi Biotec) (purity: 96.4 ± 1.0% CD11c +). . CDMo CD11c + was replated at 0.5-1.0 x 106 cells / ml in complete medium and stimulated with 100 ng / ml LPS (Sigma). After 24 h of LPS stimulation, cells were harvested for RNA isolation and immunophenotyping, and supernatants were harvested for cytokine quantification. As negative controls, parallel groups did not receive LPS.

SiRNA experiments

GILZ-targeted, silencing, screened, pre-engineered and validated siRNA (Invitrogen), with off-target prediction algorithms, was used to attenuate GILZ expression. CDMo were transfected using the Lipofectamine RNAiMAX reagent (Invitrogen). Double-stranded RNAi and lipofectamine were incubated together for 20 min, then added to CDMo cultures and incubated for 2 h at 37 ° C and 5% CO ² . Transfected CDMo were treated identically as described for CDMo / lymphocyte cocultures. CDMo was also transfected with mixed siRNA.

Immunophenotyping

Monoclonal antibodies against HLA-DR, CD80, CD83, CD3, CD86, CD14, CD11c and GILZ were included. Antibodies were obtained from Beckman-Coulter and eBioscience and used at their predetermined optimal dilutions. Apoptosis was assessed using the annexin-V apoptosis detection kit (eBioscience), with annexin-V recognizing phosphatidylserine (PS) on the surface of apoptotic cells. 7-AAD replaced PI as the cell viability stain. Cells exhibiting an annexin-V + / 7-AAD 'phenotype were classified as early apoptotic cells, and cells exhibiting an annexin-V + / 7-AAD + phenotype were classified as late apoptotic cells. Dual membrane and intracytoplasmic staining was performed using the IntraPrep fixation and permeabilization kit (Beckman-Coulter). Background staining was established with appropriate minus one fluorescence and isotype controls. Immunofluorescence was analyzed using a FACSCalibur L (BD Biosciences) within 2 h after fixation with 2% paraformaldehyde. A minimum of 10,000 events were collected for each group.

Ultra-fast quantitative PCR

RNA was isolated from CDMo CD11c + using QIAShredder columns (QIAGEN) and RNeasy Mini Kit (QIAGEN) with DNase I treatment on the column (QIAGEN). The yield and purity of the RNA were evaluated using a NanoDrop ND-1000 spectrophotometer. The cDNA was obtained using the high throughput cDNA reverse transcription kit (Applied Biosystems) in a 96-well thermal cycler (MJ Research PTC-200). TaqMan flash PCR was used to detect GILZ, CD80 and CD86 transcripts. Primers and probes were obtained as pre-engineered and validated Taqman gene expression assays (Applied Biosystems). SYBR Green flash PCR (Applied Biosystems) was used to detect transcripts of IL-12, IL-10, IL-6, TNF-alpha, and TGF-p. Primers were designed to span intron junctions using Primer3Plus. Primer melting curves were obtained to confirm a single product. HPRT-1 and GAPDH were used as reference genes. Samples were run in triplicate on a 7500 Flash PCR System (Applied Biosystems). The delta-delta C (t) procedure was used to calculate the change in times.

Cytokine quantification

The culture supernatants were analyzed in a multiple format using magnetic microspheres for IL-6, IL-8, IL-10, IL-12p70, IFN- ^y, TNF-a, RANTES, MCP-1 and MIP-1p (BioRad Laboratories ). For siRNA experiments, supernatants were analyzed with enzyme-linked immunosorbent assay (ELISA) kits for IL-10 (R&D Systems) and IL-12p70 (Enzo Life Science). All samples and standards were run in duplicate and analyzed using the LUMINEX 200 (LUMINEX) or the BioTek EL800 (BioTek).

Statistic analysis

Student's t tests were used for statistical comparisons between groups, where p values <0.05 were considered statistically significant. Differential gene expression was considered statistically significant with a> 2.5-fold change and a p value <0.05.

Results

GILZ expression is rapidly downregulated as monocytes differentiate into immature CDMo

Freshly isolated CD14 + monocytes express GILZ, but rapidly down-regulate GILZ by more than 99% as they differentiate into immature CDMo (Figure 10A). A reduction in GILZ mRNA was confirmed by a 61% decrease in GILZ protein levels (Figure 10B). GILZ down-regulation correlated with reduced expression of CD14 (monocyte-specific marker, see Zhou et al., References), and increased expression of cytoplasmic CD83 (immature CDMo marker, see Klein et al., References) . Importantly, the oDCs remained immature, expressing a low level of CD83 on the membrane (mature CD marker, see Renzo et al., References, p = 0.16). MoDCs upregulate GILZ after dexamethasone (dex) treatment in a dose-dependent manner (FIG. 10C). Treatment with dex 100 nM for 24 h was selected as a positive control for the induction of GILZ expression in CDMo (Dex-CD) (Figure 10D).

Treatment with 8-MOP or UVA alone did not achieve GILZ expression (Figure 10E). However, when the CDMo were treated with the combination of 8-MOP and UVA light (PUVA-CD), the expression of GILZ was increased 5.5-fold. GILZ induction exhibited a slow time course, peaking 24 h after treatment, and remained significantly elevated for 72 h (Figure 10F). In comparison, Dex-CD up-regulated GILZ as little as 2 h after treatment.

Immature CDMo treated with the combination of 8-MOP and UVA slightly up-regulate GILZ and assume a tolerogenic, immunosuppressive phenotype

It was then examined whether there was a dose-dependent effect of PUVA on GILZ expression. MoDCs treated with 1 J / cm2 of UVA and 100 or 200 ng / ml of 8-MOP up-regulated GILZ 2.9 and 4.4-fold, respectively (Figure 11A). A similar dose-dependent phenomenon was observed with 2 J / cm2, starting from an 8-MOP concentration of 50 ng / ml. Treatment with 0.5 J / cm2 had no effect on GILZ expression until the 8-MOP concentration reached 200 ng / ml, and treatment with 4 J / cm2 resulted in high levels of non-specific cell death (no data is displayed). The number of photoadducts formed per 106 base pairs is directly related to the product of the 8-MOP concentration and the UVA dose, see Gasparro et al., References. When the product of 8-MOP and UVA reached 100, GILZ was regulated by increment of 3 times, and as the product increased to 200 and 400, GILZ was regulated by increment of 4.8 and 8.6 times respectively (figure 11B).

The percentage of early apoptotic CD11c + cells was minimally (p> 0.05) greater than 2 J / cm2 compared to 1 J / cm2 for all 8-MOP doses tested (Figure 11C). At 2 J / cm2 and 200 ng / ml, there was an increase in the percentage of early apoptotic CD11c + cells compared to untreated CDMo (Figure 11C). The percentage of late apoptotic CD11c + cells remained less than 13% at 1 J / cm2, and less than 16% at 2 J / cm2 for all 8-MOP doses tested (Figure 11D). Furthermore, the dot plots underscore the relative resistance of the CDMo to the proapoptotic effect of increasing doses of PUVA (Figure 11E). The number of cells recovered from cultures did not differ statistically in any group treated with 1 or 2 J / cm2 (data not shown) and more than 90% of CD11c + cells were collected (range: 91.0 to 97.5 %) after treatment.

In contrast, lymphocytes show annexin-V as little as 2 h after treatment with 1 J / cm2 and 100 ng / ml (data not shown). In contrast to the CDMo treated with 100 ng / ml and 1 J / cm2 (Figure 11F), 24 h after treatment with the same dose of PUVA, the percentage of early apoptotic lymphocytes increased by 6.6% in the CDMo no 44.3% treated in PUVA-CD, and the percentage of late apoptotic lymphocytes increased from 4.5% to 33.7% (Figure 11G). Since 64.3 ± 3.2% of the lymphocytes were annexin-V + 24 h after treatment, the PUVA-treated lymphocytes are subsequently called apoptotic lymphocytes (ApoL).

The dose-dependent induction of GILZ by PUVA was correlated with a decrease in the expression of CD80, CD86 and CD83 on the cell surface (Figure 12A, 3B). The down-regulation of these markers paralleled the induction of GILZ (see Figure 11B), starting with 8-MOP concentrations of 100 ng / ml for 1 and 2 J / cm2. When the product of 8-MOP and Uv a exceeded 100, the expression of CD83, CD80 and CD86 decreased by 31%, 30% and 54% respectively, and the expression of HLA-DR increased by 38%.

CDMo exposed to apoptotic lymphocytes up-regulate GILZ and are resistant to full maturation induced by LPS

To dissect individual PUVA contributions and apoptotic cell exposure, CDMo was first co-cultured with different proportions of ApoL. GILZ was dose-dependently up-regulated by ApoL (Figure 13A). When PUVA-CD was exposed to ApoL, GILZ was expressed at higher levels than in the PUVA-CDs grown alone (Figure 13B). PUVA-CDs exposed to ApoL also expressed GILZ at higher levels than in untreated CDMo exposed to ApoL (6.7 times and 3.6 times more, respectively). There was a corresponding 1.5-fold increase in the level of GILZ protein in all groups in which the mRNA of GILZ was upregulated (Figure 13C). GILZ induction was not associated with an increase in the number of early or late apototic CD11c + cells, as there were <12% early (range 3.8 to 11.4%) and late (range 6) CD11c + cells , 3 to 11.5%) in all groups that demonstrated GILZ upregulation.

The CDMo with GILZ expression more than 2.5 times higher than the untreated CDMo were resistant to full maturation by LPS and exhibited a semi-mature tolerogenic phenotype. LPS stimulation increased CD80 expression in GILZ up-regulated CDMo to only 50% of the levels observed after LPS stimulation in untreated CDMo (Figure 13D, range: 0.48-0.57%) , and increased the expression of CD86 to only 45% of the untreated CDMo (Figure 13D, range: 0.42-0.47%). Similar results were obtained for HLA-DR and CD83 (Figure 14E, range: 47-65% and 23-57% untreated CDMo after LPS respectively). In addition, GILZ up-regulating CDMo expressed 6% of CD80 mRNA from untreated CDMo (range: 4.5-7.5%) and expressed 50% of CD86 mRNA from untreated CDMo (range : 12.4-85.1%), as evaluated by qRT-PCR.

GILZ-expressing oCDMs have a tolerogenic cytokine profile, and GILZ attenuation reduces the ratio of IL-10 to IL-12p70

Coculture supernatants were collected as described in Figure 13B. Dex-CD upregulated GILZ 4.29-fold (see Figure 13B), increased IL-10 production (Figure 14A), and decreased production of all cytokines (Figure 14B, 14C) and chemokines (Figure 14D, 14E) proinflammatory drugs tested. In comparison, PUVA-CD upregulated GILZ 2.78-fold (see Figure 13B), increased IL-10 production, and decreased the production of all pro-inflammatory cytokines and chemokines tested except TNF-α and IFN- and. Untreated PUVA-CD or CDMo, exposed to ApoL expressed GILZ at higher levels than PUVA-CD cultured alone (3.6 and 6.7 times more, respectively; see Figure 13B). These two groups increased IL-10 production and decreased the production of all pro-inflammatory cytokines and chemokines tested. Cytokine levels were confirmed at the RNA level, since the CDMo that upregulated GIL ^z also demonstrated upregulation of IL-108 mRNA times greater than the untreated CDMo (range: 5.5-11.8, p <0.01). Reductions in IL-12, TNF-a and IL-6 at the RNA level were also confirmed (data not shown). TGF-p was up-regulated by 2.5-fold in the CDMo that up-regulated GILZ (data not shown). TGF-p was not included in the multiplex analysis and was therefore only analyzed at the mRNA level.

The ratio of IL-10 to IL-12p70 is a useful indicator of tolerogenicity, since tolerogenic DCs are characterized by an increased ratio of IL-10 to IL-12p70, see Steinman et al., References). The ratio between IL-10 and IL-12p70 increased from 6.7 in untreated CDMo to 67.7 in Dex-CD. Similarly, the ratio between IL-10 and IL-12p70 increased to 38.7 in PUVA-CDs, and to 89.4 and 114.9 in untreated CDMo and PUVA-CD exposed to ApoL, respectively ( p <0.05).

To assess whether the induction of GILZ was mediating the profile of tolerogenic cytokines, CDMo was transfected with siRNA to attenuate GILZ expression. GILZ-targeted siRNA transfection reduced GILZ expression in CDMo by 68% (Figure 15A, range: 59-79%). Transfection with mixed siRNA did not significantly change GILZ expression. There were also no significant differences in the number of cells recovered from any siRNA-transfected group compared to non-transfected groups (data not shown).

Treated CDMo upregulating GILZ 2.5 times more than untreated CDMo produced higher levels of IL-10 (Figure 15B), and GILZ attenuation reduced IL-10 production by 39% (range: 34 48%, p <0.05). Treated CDMo upregulating GILZ 2.5-fold more than untreated CDMo also produced lower amounts of IL-12p70 (Figure 15C), and GILZ attenuation increased IL-12p70 production by 188% (range : 149-214%, p <0.05). The mixed siRNA treatment had no appreciable effect on IL-10 or IL-12p70 production. GILZ attenuation reduced the ratio of IL-10 to IL-12p70 that had risen after GILZ induction. Dex-CDs treated with siRNA targeting GILZ demonstrated a reduction in the ratio between IL-10 and IL-12p70 from 15.3 in non-transfected MoDCs to 3.9 in transfected Dex-CDs. In PUVA-CD, the proportion decreased from 8.4 in non-transfected CDMo to 2.9 in PUVA-CD, and in untreated CDMo and PUVA-CD exposed to ApoL, reductions in the proportion were observed from 18.1 to 7, 8 and from 28.4 to 8.3, respectively.

These results demonstrate that, like other immunosuppressive mediators, PUVA induces GILZ expression and generates tolerogenic immunosuppressive dendritic cells, characterized by low expression of the costimulatory molecules CD80 and CD86, and the CD83 maturation marker. GILZ induction is required for polarization toward a tolerogenic cytokine profile, characterized by increased IL-10 production and decreased pro-inflammatory cytokine and chemokine production, including IL-12p70. These results further implicate GILZ as the molecular switch that mediates the immunosuppressive effects of apoptotic cells.

Experiment 3 - identification of other immunostimulatory dendritic cell molecular markers Materials and procedures

Patient samples

Leukocytes were obtained from patients undergoing CSF using the UVAR XTS photopheresis system (Therakos) following the guidelines of the Yale Human Research Review Commission. Informed consent was given in accordance with the Declaration of Helsinki. Aliquots were obtained at 3 time points: before treatment (Pre-FEC), immediately after exposure to 8-MOP / ultraviolet A light (UVA) (FEC Day 0) or after 18 hours of mononuclear leukocyte incubation of treated blood (FEC Day 1) in a 1L platelet storage bag (PL-2410; Baxter).

Normal subjects

To determine whether ECF induces monocytes from healthy subjects to become DC, mononuclear leukocytes from normal subjects were examined in 2 ways.

Leukocytes subjected to leukapheresis from normal subjects (N = 3) were studied before treatment (Pre-FEC), immediately after FEC (FEC Day 0) and 18 hours after FEC (FEC Day 1). A desktop device, incorporating a UVA light source and a plastic exposure plate, allowed laboratory replication of the clinical FEC system and access to samples for RNA isolation, immunophenotyping, and parallel functional studies. Alternatively, one unit of blood from normal subjects was drawn into a transfer bag and passed through the ECF treatment apparatus in an identical manner to treated patients (N = 3). Cells obtained from the normal blood unit were used for microarray and antigen presentation assays.

Psoralen addition

As is done routinely during ECF, 8-MOP standard stock solution (Therakos) was added directly to the clinical ECF apparatus and laboratory model system. This mode of introduction allowed precise concentrations of 100-200 ng / ml throughout clinical procedures and experimentation.

I grow overnight

In ECF, it is not possible to examine phenotypic and functional changes in treated monocytes, because those cells are immediately reinfused into patients. Therefore, after ECF, cells were cultured for 18 hours (RPMI 1640/15% autologous serum) to study the activation, maturation and function of the induced monocyte genes. Before (Pre-CSF) and immediately after CSF (CSF Day 0), samples from patients and normal subjects were isolated by centrifugation on a Ficoll-Hypaque gradient. The cells were resuspended in RPMI-1640 medium (Gibco), supplemented with 7.5% AB serum, 7.5% autologous serum (Gemini Bio-Products) and cultured (those from patients) in tissue culture plates. of 6-well polystyrene at a density of 5 * 106 cells / ml and in Baxter platelet storage bags (those from normal subjects at 37 ° C, 5% CO ² ). After overnight cultivation (FEC Day 1), cells were harvested before subjecting to monocyte enrichment. To generate DC for comparative phenotypic analysis, cells were cultured in 15% RPMI 1640 serum in the presence of 1 ml of GMCSF and IL4 (25 ng / ml; R&D Systems) for 6 days.

Enrichment of the monocyte population with magnetic microspheres

In order to determine whether ECF activates the genes that lead monocytes into the dendritic cell maturation pathway, it was necessary to develop a mild monocyte negative enrichment procedure that would eliminate the contribution of lymphocytes to the transcriptome analysis while minimizing the physical or cell membrane disturbance of monocytes. Monocyte enrichment of the mononuclear cell pool was performed by a single pass through affinity columns. This negative selection procedure limited physical disturbance, while removing lymphocytes adhering to magnetic microspheres (Miltenyi Biotec), conjugated with relevant monoclonal antibodies (anti-CD4, CD8, CD19). However, enrichment of FEC Day 1 monocytes beyond 60% -80% was quite challenging, because the decreased presentation of markers on the surface of FEC-damaged lymphocytes allowed a fraction of T and B lymphocytes to escape. withholding in the columns. Repetitive passes through the affinity column, to further enhance monocyte purity, was not an option because such an approach exacerbates the physical disturbance of passively filtered monocytes. Fortunately, a series of analyzes revealed that the preferential damage of ECF to lymphocytes obviated the need for complete purification of monocytes for an accurate assessment of the level of DC gene activation. Due to their extreme sensitivity to UVA-activated 8-MOP, 99% of FEC-processed lymphocytes were apoptotic after overnight incubation (as determined by APO2-PE, trypan blue, and / or annexin-staining). fluorescein isothiocyanate (FITC) / propidium iodide). Because ECF causes global lymphocyte apoptosis, 90% -95% of viable mononuclear leukocytes in the fraction of FEC Day 1 were monocytes. This phenomenon explains the observation that the elimination of apoptotic lymphocytes with magnetic microspheres in multiple steps, carried out as follows and that provides a monocyte purity greater than 95%, does not alter the levels of gene expression observed in the cell populations studied. To achieve this comparison, we modified the monocyte purification procedure by adapting a negative selection protocol using magnetic microspheres and the EasySep magnet. Peripheral blood mononuclear cells were centrifuged at low speed (120g for 10 minutes) to remove platelets. Cells were then labeled using Monocyte Isolation Kit II (Miltenyi Biotec) following the manufacturer's procedure with the following modifications: (1) The buffer consisted of ice-cold phosphate buffered saline containing 2% autologous serum and 1 mM EDTA (ethylenediaminetetraacetic acid); (2) the blocking time was increased to 10 minutes; (3) labeling with the biotin-antibody cocktail increased to 20 minutes; and (4) the cells were washed once between labeling with the biotin-antibody cocktail and the anti-biotin microspheres. To avoid stimulating the monocytes when passing them through a column, the magnetically labeled cells were separated from the unlabeled monocytes using the EasySep magnet (StemCell Technologies). The cells, in 2 ml of buffer in a 5 ml polystyrene tube, were placed on the magnet for 10 minutes, and then the unlabeled cells were carefully poured into a new tube. This procedure was repeated 2 times, to maximize the purity of the monocytes. At this point, because the purity was still insufficient, the cells were re-labeled with the reagents from the Monocyte Isolation Kit II and placed on the EasySep magnet for an additional 10 minutes, and the unlabeled monocytes were eluted. Final purity (X = 96% + 4.5) was evaluated by flow cytometric analysis of CD14 staining.

Immunophenotyping

Monoclonal antibodies specific for monocytes and dendritic cells included: CD14 (lipopolysaccharide receptor [LPS], monocytes); CD36 (receptor for apoptotic cells, monocytes); human leukocyte antigen DR-1 (HLA-DR; major histocompatibility complex [MHC] class II molecule); CD83 (dendritic cell marker); dendritic cell cytoplasmic lysosome associated membrane protein (DC-LAMP; dendritic cell marker); and CD80 and CD86 (costimulatory molecules B7.1 and B7.2). Antibodies were obtained from Beckman Coulter and used at their predetermined optimal dilutions. Background staining with appropriate isotype controls was established, and immunofluorescence was analyzed using an FC500 flow cytometer (Beckman Coulter). Combined membrane and cytoplasm staining was performed following the manufacturer's instructions for cell fixation and permeabilization (Intraprep kit, Beckman Coulter).

Antigen presentation assay

Freshly isolated CD4 + lymphocyte populations, enriched with magnetic microspheres and exposed to antigens (2 * 106 / ml, 50 µl / well) from volunteers were added to monocytes (2 * 106 / ml, 50 µl / well) in the presence of tetanus toxoid (10 pg / ml, 100 µl / well) and RPMI 1640/15% autologous serum medium. After 5 days of culture, cells received 1 pCi of [3H] -thymidine and were incubated overnight, harvested, and counted in a Beta liquid scintillation counter (PerkinElmer). Results are presented as the mean and standard deviation of 5 replicate cultures.

MLR / CML Assay

To evaluate whether FEC processed monocytes are functionally capable of stimulating MHC class I restriction cytotoxicity by CD8 T lymphocytes, mononuclear leukocytes from 3 normal subjects were studied. One unit of anticoagulated blood, obtained recently from each of the 3 HLA-A2 positive volunteers, served as a source of dendritic / monocyte stimulator cells, before and after being processed through the clinical CEF apparatus in a manner identical to the procedure. of actual FEC. Mononuclear cell fractions were isolated from the blood immediately before processing by FEC (pre-FEC) and immediately after FEC (FEC D0). After gamma irradiation (3000 rad, cesium source) to ensure unidirectional stimulation of T lymphocytes, a serial dilution of the Pre-FEC fraction was performed in RPMI 1640/15% autologous serum, and 100 µl containing 25000 250 cells were seeded in round bottom microtiter plate wells, in 5 replicates. The FEC D0 fraction was incubated for 18 hours in large well plates and collected by scraping the wells to release adhered cells. The resuspended cells were then serially diluted and plated as before. A normal A-2 negative donor served as a source of responding CD4 and CD8 T cells, purified by positive selection on Miltenyi magnetic microsphere columns (average purity: 98%). Responding T lymphocytes (50,000 / well in 100 µl) were then added to wells containing Pre-FEC or FEC-D0 stimulators, and the plates were cultured for 7 days at 37 ° C in a CO ² incubator. For the target cells, the TB A-2 positive hybridoma lymphoblast line, 174 x CWM.T1, was labeled with 51 Cr and added to the MLR cultures at 104 cells / well. After 4 hours of incubation, the plates were centrifuged and 100 µl of supernatant was removed from each well for counting in a gamma counter. The "percent specific lysis" was defined as 100 times the following fraction:

mean cpm (sample) - mean cpm (T lymphocytes only)

mean cpm (maximum detergent release) - mean cpm (T lymphocytes only)

RNA isolation and microarray hybridization

Total RNA was isolated using RNeasy Mini Kit columns with DNase I treatment on the column (QIAGEN). The yield and purity of the RNA were measured using the NanoDrop ND-1000 spectrophotometer and the Agilent 2100 bioanalyzer. The fragmented cRNAs were hybridized on human Affymetrix HG U133 Plus 2.0 chips, and the WM Keck Resource Laboratory at Yale University performed a screening for approximately 47400 human genes and ESTs. Microarray results are available from Gene Expression Omnibus under accession number GSE23604.

Data analysis

Raw and unnormalized data generated by the Affymetrix GeneChip Operating Software Version 1.2 software (GCOS 1.2; Affymetrix) were analyzed using the GeneSpring 7.2 software (Agilent Technologies-Silicon Genetics). The data was normalized using the Robust Multi-Array. Only sets of probes with a minimal fold change of> 2.0 combined with an average signal intensity of 500 or more in leukapheresis or treated samples were included in the analysis. Differential gene expression was considered as a> 2-fold change and P <0.05. Principal component analysis (PCA) of the induced transcriptomes was performed by standard methodology. Participation in the signal transduction pathway was identified with the MetaCore Version 1.0 (GeneGo) software.

Ultra-fast quantitative PCR

Microarray expression of selected genes was confirmed in aliquots of the same RNA samples, using ultra-fast quantitative polymerase chain reaction (PCR). Reverse transcription of RNA to cDNA was performed using the high capacity cDNA reverse transcription kit (Applied Biosystems). Reverse transcription was performed in a 96-well thermal cycler (MJ Research PTC-200) under the following conditions: 25 ° C, 10 minutes, 37 ° C, 120 minutes, 85 ° C, 5 seconds. TaqMan flash PCR was used to detect DC-LAMP, CCR7, CD80, CD86 and CD14 transcripts. The primers and probes for each sequence were obtained as Taqman gene expression catalog assays (Applied Biosystems). HPRT1 was used as the reference gene.

Results

ECF induces large changes in individual gene expressions

The ECF stimulation of the activation of individual genes in monocytes was expressed as the ratio between Day 1 CSF and Pre-CSF expression for the relevant gene. To avoid inadvertent gene induction during monocyte enrichment, a negative column purification procedure was used, whereby lymphocytes were retained, and monocytes were passively filtered. The results revealed that FEC-processed monocytes from normal patients and subjects remain viable enough to reproducibly express a common transcriptome signature.

Genes were considered to be significantly up or down regulated by FEC if the fold change was> 2 and significance was P <0.05 compared to Pre-FEC. The levels of RNA transcripts of approximately 3000 genes underwent significant changes in each group of patients and in normal subjects (Table 2). Overall, 1129 common genes had been up-or down-regulated by ECF-processed monocytes from LCLT and GVHD patients and from normal subjects, indicating homogeneity in ECF-induced gene activation.

Table 2: Number of genes of monocytes with altered expression after ECF.

The increased expression of numerous genes associated with dendritic cell differentiation, adhesion, and function (Table 3) further supports ECF stimulation of monocyte entry into this pathway.

Table 3: ECF-enhanced expression of CD marker genes, ratio * of post-ECF / pre-ECF levels

Table 1 shows other genes, whose expression was increased and that can be considered molecular markers of immunostimulating dendritic cells.

As expected during maturation of monocytes to dendritic cells, CD14 (monocyte marker) expression decreased, as assessed by measuring the mean fluorescence intensity in monocyte populations from all patients and normal subjects, after culture during the night of monocytes processed by FEC. This result was confirmed in the RT-PCR studies of the patients' post-CSF cells (results not shown). Other factors, whose expression was reduced indicating maturation of monocytes to dendritic cells are shown in table 4.

Table 4: Reduced expression by ECF of monocyte marker genes, ratio * of post-ECF / pre-ECF levels

Other factors, whose expression was reduced and therefore indicate maturation of monocytes to immunosuppressive dendritic cells, are shown in Table 5.

Table 5: ECF-enhanced expression of genes associated with immunosuppression, ratio * of post-ECF / pre-ECF levels.

Experiment 4 - Markers of surface molecules and functional mediators of immunostimulating DC.

Another FEC-induced dendritic cell transcriptome analysis was performed to identify a subset of surface molecule gene products as functional immunostimulatory dendritic cell markers and mediators. 466 dendritic cell FEC-induced upregulated genes and approximately 2000 known or suspected full-length human transmembrane genes were cross-referenced to identify 87 common surface proteins.

Materials and procedures

Obtaining leukocytes and platelets

All samples were obtained from young, healthy subjects who were not taking medications, including acetylsalicylic acid, which is known to influence platelet function. Samples were obtained following the guidelines of the Yale Human Research Review Commission and informed consent was given in accordance with the Declaration of Helsinki. Peripheral blood samples were drawn through a gauge needle 19 from the antecubital vein with syringes containing heparin, then applied to Ficoll-Hypaque (Gallard-Schlessinger, Carle Place, NY). After centrifugation at 180 g, the interface containing the fraction of mononuclear leukocytes was collected and washed twice in HBSS, then resuspended in RPMI-1640 medium (GIBCO) at a final concentration of 5 x 106 mononuclear cells / ml. Cells were used within one hour of being obtained.

Platelet Rich Plasma Preparation

Whole blood was centrifuged at 150g for 15 minutes at room temperature. The platelet rich plasma (PRP) layer was collected and centrifuged at 900g for 5 minutes, and the platelet pellet was resuspended in RPMI 1640 at the desired concentration.

Plate Preparation

Plating was performed using a Glycotech system (Glycotech, Rockville, MD). This system consisted of a volumetric flow path measuring 20,000 x 10,000 x 254 microns (length x width x height). The lower plate of this system consisted of a 15 mm Petri dish (BD Biosciences, Durham, NC) separated by a gasket and connected under vacuum to an acrylic flow platform, which formed the upper plate. For precoating with platelets, before assembling the flow chamber, 20 drops of the desired concentration of PRP were placed in the center of the Petri dish and the platelets were allowed to stand for 20 minutes at room temperature. The Petri dish was washed twice with 2 ml of RPMI and then the flow chamber was assembled.

I grow overnight

When overnight culture was necessary, cells were centrifuged and resuspended in RPMI-1640 medium (GIBCO), supplemented with 15% AB serum (Gemini Bio-Products) at a final concentration of 5 x 106 cells / ml. Cells were grown overnight for 18 hours in 12-well polystyrene tissue culture plates (2 ml per well) at 37 ° C in 5% CO ² .

Immunophenotyping

Monoclonal antibodies for immunophenotyping included CD14 (LPS receptor; monocytes), CD11c (integrin subunit; monocytes and CDs), HLA-Dr (MHC class II molecule), CD83 (CD marker), CD62p (P-selectin ; activated platelets), and CD61 (integrin subunit; platelets). Antibodies were obtained from Beckman Coulter (CD14, CD11c, HlADR, CD83) or Sigma (CD62p, Cd61) and used at their predetermined optimal dilutions. Background staining with appropriate isotype controls was established, and immunofluorescence was analyzed using an FC500 flow cytometer (Beckman Coulter). Two-color membrane staining was performed by adding the predetermined optimal concentrations of both antibodies directly conjugated to FITC or PE and incubating for 20 minutes at 4 ° C, followed by washing to remove unbound antibodies. Combined membrane and cytoplasm staining was performed following the manufacturer's instructions for cell fixation and permeabilization (Intraprep kit, Beckman Coulter).

Results

Plaque and / or PBMC D1 populations showed significant upregulation of the analyzed surface expression of SIRPa, ICAM1, CXCL16, LIGHT, PLAUR (CD87, plasminogen activator, urokinase receptor), MSR1, Neu1 (sialidase) , CD137L and CATB (CTSB, cathepsin B).

Experiment 5 - Determination of molecular marker expression and complexity by FSC / SSC after passing monocytes through the flow chamber

Materials and procedures

Monocytes were passed through a device depicted in Figure 19. Briefly, a blood sample was centrifuged at low speed through a Ficoll gradient to obtain, for example, 8 ml of sample with a mononuclear cell concentration of peripheral blood (PBMC) of, for example, 1010 cells / ml. The chamber had been previously coated with platelets. The sample was passed through the chamber at approximately 0.028 Pa. The chamber was then washed with approximately 3 ml of RPMI at 0.028 Pa. A second wash was performed with 30-55 ml of RPMI at approximately 1.2 Pa. Collected activated monocytes were pooled, incubated for one day and used for further analyzes (PBMC D1 PP). As a control, PBMC that were not passed through the device and incubated for one day (PBMC D1). As an additional control, rapid immature DCs were obtained by directly culturing PBMC in the presence of GM-CSF and IL-4 (fast immature DCs). Furthermore, PBMC were analyzed directly after harvesting through a Ficoll gradient (fresh PBMC (Ficoll)).

The cells and controls were then analyzed for the expression of HLA-DR, CD86, ICAM-1 and PLAUR. They were re-analyzed for complexity by FSC / SSC. The HLA-DR results are depicted in Figure 20 and the FSC / SSC complexity results in Figures 21 and 22. A summary is shown in Figure 23.

Results

The results show that cells subjected to centrifugation through a Ficoll gradient already appear to experience sufficient physical forces to begin to differentiate as evident by incubating these cells for one day (PBMC D1). However, activation and differentiation is more pronounced after passing the plate through the device (PDMC D1 PP). Dendritic cells obtained by methods according to the invention in the absence of, for example, 8-MOP and UV-A, furthermore have a more complex and distinctive pattern than immature fast DCs obtained with cytokine cocktails.

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Claims (14)

A method for inducing the differentiation of monocytes contained in an extracorporeal amount of a blood sample from a mammalian subject into immunostimulatory autologous dendritic cells, said method comprising at least the steps of: a) subjecting said extracorporeal quantity of said blood sample from a mammalian subject to a shear stress passing through a flow chamber that allows a fixed or adjustable flow rate adjustment; b) wherein the procedure is performed in the absence of photoactivatable agents such as 8-MOP and UVA c) wherein the procedure is performed without the need to add a molecular cocktail comprising cytokines; and d) in which the differentiation of monocytes from said blood sample into immunostimulating autologous dendritic cells is identified by the increased expression of at least one molecular marker HLA-DR, CD83, CD86, ICAM1 or PLAUR and not increased expression of GILZ when comparing immunostimulating autologous dendritic cells with monocytes within the extracorporeal amount of a mammalian subject blood sample. 2. Method according to claim 1, wherein the differentiation of monocytes of said blood sample into immunostimulatory autologous dendritic cells as indicated in step d) is identified by the increased expression of at least 5 molecular markers HLA-DR , CD83, CD86, ICAM1 and PLAUR. Method according to claim 1 or 2, wherein said device additionally allows the adjustment of at least one parameter selected from the group comprising temperature and exposure to light. 4. Process according to any of claims 1 to 3, wherein said monocytes are activated and induced to differentiate into immunostimulating autologous dendritic cells through interaction with activated platelets and / or plasma components. 5. Method according to any of claims 1 to 4, in which the activation of such monocytes and the differentiation into immunostimulating autologous dendritic cells can be influenced by the design and dimensions of the flow chamber, the flow rate at which the monocytes pass through the flow chamber, the temperature at which monocytes, platelets, platelet-derived factors and / or plasma components are passed through the flow chamber, exposure of monocytes to light, the order in which monocytes, platelets, platelet-derived factors, and / or plasma components are passed through the flow chamber, the density with which the plasma components coat the surfaces of the flow chamber, the density with which platelets and / or platelet-derived factors adhere to the surfaces, and / or the plasma components of the flow chamber, and / or the density with which monocytes adhere to platelets and / or platelet-derived factors and / or plasma components attached to the surfaces of the flow chamber. 6. Process according to any of claims 1 to 5, i) wherein said procedure comprises at least the steps of: a) applying said extracorporeal amount of said mammalian subject blood sample comprising at least monocytes to a device, which is configured to provide a flow chamber through which said extracorporeal amount of said subject blood sample can be passed mammal, b) activating platelets, which may be comprised within said extracorporeal amount of said mammalian subject blood or which may be provided separately from said mammalian subject blood sample comprising at least monocytes, c) treating said extracorporeal amount of said mammalian subject blood sample comprising at least monocytes in said device by applying a physical force to the monocytes contained within said extracorporeal amount of said mammalian subject blood sample, such that said monocytes are they activate and are induced to differentiate into immunostimulatory autologous dendritic cells by binding to said activated platelets obtained in step b); or ii) wherein said process comprises at least the steps of: a) applying said extracorporeal amount of said mammalian subject blood sample comprising at least monocytes to a device, which is configured to provide a flow chamber through which said extracorporeal amount of said subject blood sample can be passed mammal, b) passing plasma components, which may be comprised within said extracorporeal amount of said mammalian subject blood sample or which may be provided separately from said mammalian subject blood sample, c) treating said extracorporeal amount of said mammalian subject blood sample comprising at least monocytes in said device by applying a physical force to the monocytes contained within said extracorporeal amount of said mammalian subject blood sample such that said monocytes are activated and they are induced to differentiate into immunostimulating autologous dendritic cells by binding to said plasma components obtained in step b); or iii) wherein said procedure comprises at least the steps of: a) applying said extracorporeal amount of said mammalian subject blood sample comprising at least monocytes to a device, which is configured to provide a flow chamber through which said extracorporeal amount of said subject blood sample can be passed mammal, b) passing plasma components, which may be comprised within said extracorporeal amount of said mammalian subject blood or which may be provided separately from said mammalian subject blood sample, c) activating platelets, which may be comprised within said extracorporeal amount of said mammalian subject blood sample or which may be provided separately from said mammalian subject blood sample comprising at least monocytes, d) treating said extracorporeal amount of said mammalian subject blood comprising at least monocytes in said device by applying a physical force to the monocytes contained within said extracorporeal amount of said mammalian subject blood sample such that said monocytes are activated and activated. they induce differentiation into immunostimulatory autologous dendritic cells by binding to said activated platelets and / or plasma components obtained in steps b) and c). 7. The method according to claim 6, wherein said extracorporeal amount of said mammalian subject blood sample has not been obtained by apheresis; in which preferably: i) said extracorporeal amount of said mammalian subject blood sample is between about 10 ml and about 500 ml of extracorporeal whole blood from said mammalian subject; me ii) said extracorporeal amount of said mammalian subject blood sample is obtained by isolating leukocytes from about 10 ml to about 500 ml of extracorporeal whole blood from said mammalian subject; me iii) said extracorporeal amount of said mammalian subject blood sample is obtained by isolating buffy coats from about 10 ml to about 500 ml of extracorporeal whole blood from said mammalian subject; me iv) said extracorporeal amount of said mammalian blood sample does not comprise plasma components; me v) said extracorporeal amount of said mammalian subject blood sample does not comprise platelets, wherein preferably said platelets have been separated from said extracorporeal amount of said mammalian subject blood before said extracorporeal amount of said mammalian subject blood is applied to that device. 8. Method of claim 7, wherein said extracorporeal quantity of said mammalian subject blood has been obtained by apheresis, wherein preferably: i) said extracorporeal quantity of said mammalian blood is obtained by isolating leukocytes by apheresis; me ii) said extracorporeal quantity of said mammalian subject blood is obtained by isolating buffy coats by apheresis; me iii) said extracorporeal amount of said mammalian blood does not comprise plasma components; me iv) said extracorporeal amount of said mammalian subject blood does not comprise platelets, wherein preferably said platelets have been separated from said extracorporeal amount of said mammalian subject blood before said extracorporeal amount of said mammalian subject blood is applied to said device. The method of any of claims 7 to 8, wherein said flow chamber has dimensions of about 1 pm to about 400 pm in height and from about 1 pm to about 400 pm in width; wherein preferably said flow chamber has dimensions from about 5 pm to and including about 300 pm in height and from about 5 pm to and including about 300 pm in width; wherein more preferably said flow chamber has dimensions from about 10 pm to and including about 250 pm in height and from about 10 pm to and including about 250 pm in width; wherein even more preferably said flow chamber has dimensions of about 50 µm up to and including about 200 µm in height and from about 50 µm up to and including about 200 µm in width; and wherein even more preferably said flow chamber has dimensions of from about 50 µm to and including about 100 µm in height and from about 50 µm to and including about 100 µm in width. The method of claim 9, wherein said flow chamber is configured to receive a volume of between about 1 ml and about 50 ml of said extracorporeal amount of said mammalian subject blood sample. 11. The method of any of claims 3 to 10, wherein i) the material of said flow chamber is not plastic; and wherein preferably said non-plastic material is glass; or ii) the material of said flow chamber is plastic; wherein preferably said plastic material is selected from the group consisting of acrylics, polycarbonate, polyetherimide, polysulfone, polyphenylsulfone, styrenes, polyurethane, polyethylene, Teflon, or any other appropriate medical grade plastic; and / or iii) said flow chamber is configured to allow transmittance of light, wherein preferably said flow chamber is configured to allow transmittance of UV light. 12. The method of any of claims 4 to 11, wherein the activation of said platelets is achieved by arranging plasma components, which are comprised within said extracorporeal amount of said blood sample from a mammalian subject, on the surface of said chamber of flow so that at least some of said platelets can interact with said plasma components and remain immobilized on the surface of said flow chamber; in which preferably: i) the activation of said platelets is achieved by arranging proteins selected from the group comprising fibrinogen, fibronectin and the gamma component of fibrinogen on the surface of said flow chamber so that at least some of said platelets can interact with said proteins and remain immobilized on the surface of said flow chamber; and wherein most preferably the activation of said platelets is achieved by arranging fibrinogen on the surface of said flow chamber so that at least some of said platelets can interact with said fibronectin and remain immobilized on the surface of said flow chamber; me ii) said platelets are passed through said flow chamber under a shear stress of 0.1 to 10.0 dynes / cm2, preferably in a range of about 0.1 to about 2.0 dynes / cm2; me iii) platelet activation can be controlled by the expression of P-selectin and / or aIIb-p3 integrin. 13. The method of any of claims 4 to 12, wherein i) said monocytes are passed through said flow chamber at a flow rate of about 10 ml / minute to about 200 ml / minute to produce a shear stress of about 0.1 to about 20.0 dynes / cm2; me ii) said monocytes are activated and induced to differentiate into immunostimulating autologous dendritic cells by passing said monocytes through said flow chamber under a shear stress of approximately 0.1 to approximately 10.0 dynes / cm2, preferably a tension of approximately 0.1 to about 1.0 dynes / cm2 so that said monocytes can bind to said activated platelets. The method of any of claims 1 to 13 for obtaining functionally and maturately synchronized autologous immunostimulatory dendritic cells individual specific.